Background
The geographical and socio-economic distribution of malaria overlaps with areas in which a number of helminth parasites are also endemic. It is the norm in these areas for co-infection to occur and a growing body of literature reflects this [
1‐
12]. The influence of co-infection on the immune response may result in either exacerbation or amelioration of disease [
13‐
15]. It is therefore crucial to understand the host-parasite relationship in the context of multiple infections, if vaccine design and drug administration programmes are to be managed effectively [
16]. Animal models accurately reflect many pathological aspects of malaria-helminth co-infection with regard to impact on disease outcome and also provide the opportunity to further examine immunological mechanisms in detail [
17‐
20].
We previously undertook an investigation to assess the impact of a pre-existing chronic nematode infection on malaria-related pathology, utilising the rodent malaria
Plasmodium chabaudi chabaudi (Pcc) and the rodent filarial nematode
Litomosoides sigmodontis (Ls)[
21]. We found that co-infected mice (
Pcc-Ls), particularly those that did not have blood microfilaremia, had exacerbated immunopathology. This was associated with increased interferon-gamma (IFN-γ) responsiveness but was independent of
Pcc parasitemia [
21]. One of the primary objectives in our previous malaria-nematode co-infection studies was to gather antigen-specific T-cell data to determine whether nematode infection could alter the cytokine bias of the
Pcc-specific T lymphocyte response towards Th1 and conversely, whether a potent Th1 response could alter the Th2 bias of the nematode-specific response.
Cytokine production by antigen specific T-cells can be difficult to assess during malaria, due to immune suppression associated with the peak of infection and apoptosis of splenocytes [
22]. Additionally, the complex nature of the target antigen (
Pcc-infected red blood cells) is a further complicating factor. Thus, gathering antigen-specific T-cell data remains a technical challenge of studying immunity to malaria particularly in human studies where there is the additional challenge of obtaining and maintaining lymphocytes in the field.
Here we focus on the dissection and interpretation of parasite antigen-specific antibody responses as an alternative to T-cell analysis. Antibodies of the IgG2a isotype are mainly produced by B cells in response to IFN-γ in mice [
23‐
25] whereas the Th2 cytokine IL-4 switches B cells to produce IgG1 [
24,
26]. Although the generation of IgG1 as a marker for Th2 cells is less definitive than IgG2a as a marker of a Th1-type response, the ratio of IgG1 to IgG2a provides a powerful indicator of immune bias [
27‐
30]. Measurement of antibodies can also be achieved with smaller sample volumes and poses fewer technical challenges than T-cell recall assays. Furthermore, antibody analysis can provide information on the fuller history of infection as it reflects cumulative immunological activity, whereas cytokine responses of T-cells are an
ex-vivo 'snapshot' that can more readily be altered by changes in the timing of sampling both
in vivo and
in vitro. Antibody analyses of co-infected animals might therefore provide evidence of overall Th1-Th2 cell cross-regulation even when cytokine analyses may not.
In addition to their use as indicators of cytokine bias during infection, antibody isotypes have direct functional relevance to disease severity in helminth-malaria co-infection. Antibodies are absolutely required for the ultimate clearance of malaria parasites [
31]. In mice, antibodies of the cytophilic isotype IgG2a have been shown to recognise infected erythrocytes [
32] and facilitate their destruction by phagocytes [
33]. Similarly, in humans IgG1 and IgG3 are associated with enhanced parasite clearance [
34]. If helminth co-infection alters antibody class-switching and consequently the production of malaria-specific cytophilic antibodies then the resolution of malaria infection may be affected. Indeed, co-infection with the gastro-intestinal nematode
Heligomosoides polygyrus reduced
Pcc- specific IgG2a responses and resulted in exacerbated malaria parasitemia [
18]. There are also important implications for vaccine efficacy and administration. For example, immunisation that protected mice from malaria failed to do so in mice that also harboured a nematode infection [
35].
In this study, the characterisation of antibody isotype responses as an indicator of cytokine bias during co-infection has proved unexpectedly challenging due to the production of cross-reactive antibodies induced by single-species infection. To establish the real effect of co-infection on the Th1/Th2 immune bias from non-specific reactivity to antigen we needed to determine how robust the cross-reactive responses were in comparison to the antigen-specific. We demonstrate that a combination of calculating antibody titre, from a dilution series of test sera, and periodate treatment of the parasite antigens can control for most cross-reactivity. The magnitude and robustness of some cross-reactivity, however merits further investigation to explore the potential function of these responses during co-infection.
Methods
Hosts, parasites and experimental infection
Specific pathogen free, 8-10 week old female BALB/c mice (Harlan, UK) were maintained in individually ventilated cages on diet 41b ad lib in a 12 h:12 h light-dark cycle. All experiments were carried out in accordance with the animals (Scientific Procedures) Act 1986, and were approved by the UK Home Office inspectorate and institutional review committee.
Pcc clone AS was originally isolated from thicket rats (
Thamnomys rutilans) and was cloned by serial dilution and passage [
36]. Parasites were recovered from frozen blood stabilates by passage through donor mice. Experimental parasite inoculations were prepared from donor mice by diluting blood in calf serum solution (50% heat-inactivated foetal calf serum, 50% Ringer's solution [27 mM KCl, 27 mM CaCl2, 0.15 M NaCl, 20 units heparin per mouse]). Each mouse received 0.1 ml of inoculum intraperitoneally (i.p) corresponding to an infective dose of 1 × 10
6 or 1 × 10
5 parasitized red blood cells (RBC), depending on the experiment. An inoculum of naïve RBC was given as a control for erythrocyte proteins.
The filarial nematode
Ls was maintained by cyclical passage between gerbils (
Meriones unguiculatus) and mites (
Ornithonyssus bacoti) as described previously [
37]. Infection was initiated by subcutaneous (s.c) injection of 25 infective (L3) larvae. For co-infection experiments in which the influence of malaria on chronic nematode infection was addressed, 1 × 10
6Pcc parasitized RBC were introduced i.p on Day 60 of an established
Ls infection and mice were sacrificed on day 20 post-
Pcc infection, as described previously [
21]. Whole blood was collected from the brachial artery and serum recovered after clotting at room temperature.
Nb worms were maintained by serial passage through Sprague-Dawley rats. L3 larvae were obtained by culturing the faeces of infected rats at 26°C for a minimum of 5 days [
38]. For acute nematode-malaria co-infection, infection was initiated by s.c injection of 200 infective (L3) larvae on the same day that
Pcc was introduced by inoculation i.p of 1 × 10
5 parasitized RBC. Mice were sacrificed on Day 20 post-infection under terminal anaesthesia. Whole blood was collected from the brachial artery and was separated using Sera Sieve (Hughes & Hughes Ltd).
Antigens
Two malaria antigens were used in this study: a recombinant protein and a crude antigen homogenate prepared from parasitized erythrocytes. The recombinant Merozoite Surface Protein-1
19 (MSP-1
19) was originally sequenced, cloned and expressed from
Pcc AS clone, as described previously [
39]. In brief, the MSP-1
19 nucleotide sequence was inserted into
Pichia pastoris vector pIC9K and protein expression carried out in
Pichia pastoris strain SMD1169. This antigen was used in ELISA at a concentration of 1 μg/ml.
The crude malaria homogenate - lysed Pcc parasitized red blood cell extract (pRBC) - was prepared from whole blood of mice with a parasitemia in excess of 20%. Mice were bled by cardiac puncture with a heparinised syringe and blood stored at -80°C prior to 3 rounds of freeze-thaw to lyse the parasitized red blood cells. The lysed cells were sonicated, on ice, twice for 30 sec at 10 Amp and centrifuged at 16060 g for 10 min. The supernatant was stored at -80°C. Similarly, a naïve red blood cell extract (nRBC) was prepared as a control for RBC proteins; responses to this antigen amongst infected mice were indistinguishable from naïve (data not shown). In the Ls experiments this antigen was used in ELISA at 0.5 μg/ml and in the Nb experiments at 5 μg/ml.
Ls and Nb extracts (LsA and NbA) were prepared by homogenisation of adult nematodes in PBS. The somatic extracts were centrifuged at 1000 g for 20 mins and the pellet discarded. The extract was stored at -20°C. LsA was used in ELISA at 0.5 μg/ml and NbA at 5 μg/ml.
Antibody detection
ELISA was used to measure antigen-specific IgG antibodies in the serum of nematode-infected, Pcc- infected or co-infected mice. In the Pcc-Ls study, sera were added in a serial dilution 1/100 - 1/400 and a dilution was then chosen whereby all samples fell in the linear range of the curve; for IgG1, a dilution of 1/200 and for IgG2a 1/100. For the subsequent Pcc-Nb study, serum samples were added in a serial dilution 1/50 - 1/819200. Antibody titres were calculated as the reciprocal of the greatest dilution at which optical density (O.D) was greater than the mean plus 3 standard deviations of the O.D values observed for control mouse sera at 1/200 dilution.
Antibody responses to MSP-119, pRBC, NbA or LsA were determined for IgG isotypes IgG1, IgG2a, and IgG3. 96 well maxisorp immunoplates (Nunc) were coated at 4°C overnight with either recombinant or crude antigens at the concentrations indicated (see Antigens section) in 0.06 M carbonate buffer (0.04 M NaHCO3, 0.02 M NaCO3, pH9.6) in a final volume of 50 μl per well. Non-specific binding was blocked with 5% FCS in carbonate buffer (200 μl/well) for 2 hours at 37°C. Wells were washed three times in Tris buffered saline with 0.1% Tween (TBST) after each step. Serum samples were added in serial dilutions as indicated using TBST as a diluent, in a final volume of 75 μl per well and incubated for 2 hours at 37°C. Isotype specific detection antibodies were diluted in TBST in a final volume of 50 μl per well. For IgG1, HRP conjugated goat anti-mouse IgG1 (Southern Biotech 1070-05) was used at 1/6000, HRP conjugated goat anti-mouse IgG2a (Southern Biotech 1080-05) at 1/4000 and HRP conjugated goat anti-mouse IgG3 (Southern Biotech 1100-05) was used at 1/1000. Plates were incubated for 1 hour at 37°C. An additional wash in distilled water was carried out before developing with ABTS peroxide substrate (Insight Biotechnology), 100 μl per well, at room temperature for 20 minutes. O.D was read at 405 nm using a spectrophotometer.
Polyclonal IgE levels were determined by sandwich ELISA. 96 well maxisorp immunoplates (Nunc) were coated overnight at 4°C with 100 μl of IgE capture antibody (2 μg/ml; clone R35-72 Pharmingen) diluted in carbonate buffer. Plates were blocked with 5% non-fat skimmed milk in carbonate buffer for 2 hr at 37°C. Plates were washed 5 × in TBST before addition of sera at 1/10 and 1/20 dilutions in a final volume of 50 μl/well and left overnight at 4°C. For the standard curve two- fold serial dilutions of purified mouse IgE, κ monoclonal isotype standard (Pharmingen) were used. After 5 washes in TBST, 100 μl of biotinylated detection antibody (2 μg/ml; clone R35-118 Pharmingen) diluted in TBST with 5% FCS was added and plates left at 37°C for 90 mins. Plates were washed 5 × in TBST prior to incubation with ExtrAvidin peroxidase (SIGMA), diluted 1:8000 in TBST with 0.5% FCS, for 30 mins at 37°C. After a final wash in distilled water, plates were developed with 100 μl TMB microwell peroxidase substrate system (Insight Biotechnology Ltd) and read at 650 nm.
In order to determine the extent to which carbohydrate or protein moieties contributed to the antibody response the antigens were pre-treated with periodate. Antigen-specific IgG1, IgG2a and IgG3 antibodies were measured in response to antigens treated with periodate. The ELISA was carried out as detailed for the Nb co-infection experiment with untreated antigens but the following additional steps were included after blocking with 5% FCS: carbonate buffer, prior to sample addition. TBST wash (×3) was followed by the addition of 10 mM sodium (meta) periodate diluted in 50 mM sodium acetate in a final volume of 100 μl/well. Plates were incubated at 37°C for 1 hour and then washed in 50 mM sodium acetate. To stop the activity of periodate, 100 μl of 50 mM sodium borohydride solution was added to each well.
Statistical Analysis
General linear statistical models allowed us to frame and test questions such that we could determine whether differences in infection status and/or presence of carbohydrate antigen explained the observed variation in antibody responses. For more detailed explanation of the statistical methods employed see Grafen and Hails [
40]. Infection status and treatment with periodate (or not) were included as categorical factors and their ability to predict antibody response was formally evaluated via Analysis of Variance (ANOVA). The serial dilution of sera in an ELISA produces ordinal data, which were log
10 transformed prior to analysis to ensure the data were approximately normally distributed, in accordance with the requirements of linear models. Analyses were carried out using the statistical package JMP 5.1 (SAS). The maximal model was fitted first and minimal models were obtained by sequentially removing non-significant terms (P-value > 0.05), beginning with interactions. Finally, whenever a factor was significant (
P < 0.05), an All Pairs Tukey post-hoc test was carried out to identify which groups of mice differed significantly in antibody induction, with respect to infection status or periodate treatment.
Discussion
Antibody analysis should be able to provide critical information on changes in cytokine bias due to co-infection. This is particularly important in human studies where serum may be the only reagent available for immunological analysis. Whilst we acknowledge that there is a need to confirm the relationship between splenic or serum cytokines and antibody responses in co-infection if this strategy is to be used in human studies, our focus is on the interpretation of antigen-specific Th1/Th2 bias based on antibody isotype, which was complicated by cross-reactivity in the two co-infection models studied here. It is worth noting that cross-reactive responses were observed regardless of whether recombinant or crude antigens were used. We primarily address technical strategies that will enable us, and others, to draw conclusions regarding the influence of a co-infecting parasite on immune bias using serum antibodies. However, the functional implications of cross-reactive responses are also discussed.
Murine models that aim to dissect the real effect of a co-infecting parasite on immune bias must use large numbers of animals to detect significant differences in antigen-specific responses between single and dual infection. Thus for antibody analysis of the large sample size (see legend Fig
1 for details) in our
Pcc-Ls study of co-infection we chose a fixed serum concentration, previously determined to fall within the linear range of the dilution curve. Although this saved time and reagents, in retrospect, it provided insufficient information for our purposes: it did not allow us to distinguish the relative strengths of cross-reactive versus antigen-specific responses.
When antibody titres were calculated in the
Pcc-Nb study, we were able to determine whether apparent alterations in antibody isotype profile on co-infection were due to actual changes in parasite-specific responses or reflected a cross-reactive response. For example, determining that cross-reactive IgG2a antibody titres in
Nb mice (X
4 in Fig
2Bi) were significantly lower than the antigen-specific response of
Pcc mice meant that cross-reactivity was unlikely to influence the titre observed in
Pcc-Nb mice. This allowed us to conclude that the reduction in Th1 type antibody in
Pcc-Nb mice was probably due to suppression of
Pcc-specific Th1 responses by nematode infection. Further to this, had we not calculated titre and relied on optical density data derived from a single dilution of sera we may not have observed the difference between
Pcc and
Pcc-Nb mice and thus incorrectly concluded that there was no effect of co-infection on Th1 responses. Similarly, analysis of antibody titre enabled us to detect the reduction in anti-NbA IgG1 antibody in
Pcc-Nb mice (Fig
2Aiii), which suggests a
Pcc-mediated bias toward a Th1 cell response. In other cases, cross-reactivity was observed even at high dilutions with
Pcc mice achieving IgG2a titres equivalent to or greater than
Nb mice (X
6 in Fig
2Biii). In this case, calculation of titre did not help to unravel potential cytokine influences and the enhanced IgG2a response in co-infected mice may be due to increased Th1 cytokines during co-infection and/or the presence of cross-reactive antibody (Fig
2Biii).
Nematode surface antigens and the excretory/secretory products from these parasites are heavily glycosylated [
59]. Similarly,
Plasmodium species express glycoconjugates on their surface and have abundant glycophosphatidylinositol anchors [
60]. In other co-infection systems cross-reactive epitopes have been shown to derive from carbohydrate structures [
61]. The sensitivity of the carbohydrate component of an antigen to periodate treatment [
59] has been beneficial in interpreting our results. In particular, treatment of
Pcc antigens demonstrated that cross-reactive nematode-induced IgG3 responses were largely attributed to the carbohydrate component. Interestingly, periodate treatment also reduced apparent cross-reactivity by exposure of protein epitopes, previously masked by carbohydrate, which enhanced the detection of antibodies from mice that had been exposed to the antigen during infection (e.g., anti-pRBC in Fig
3Bii). This allowed us to conclude that levels of anti-pRBC IgG2a antibody in
Pcc-Nb mice are solely induced by the
Pcc parasite. Further to this, detection of cross-reactive protein-specific antibodies enabled responses, previously indistinguishable in magnitude between singly-infected groups, to be differentiated. For example, periodate treatment of NbA enhanced detection of
Pcc induced cross-reactive IgG2a antibodies (X
8 in Fig
3Biii) whilst the antigen-specific response of
Nb mice was ablated. This indicates that the level of
Nb-specific IgG2a observed in
Pcc-Nb mice is due to
Pcc driving a cross-reactive IgG2a Th1 type response.
We have demonstrated that the use of serial dilutions and periodate treatment of the parasite antigens can help overcome cross-reactivity for the purposes of analysing and interpreting Th1/Th2 cell immune bias. However, some 'true' cross-reactivity remained (i.e.
Pcc-induced IgG2a responses to NbA (Fig
2Biii/Fig
3Biii)), and the induction of these antibodies has important implications with regard to biological function. For example the immune responses to nematode infection are typically characterised by a Th2 type (IgG1) response, as we observed for
Nb-induced responses to the nematode antigen (NbA). The propensity for
Pcc mice to induce atypical IgG2a antibody isotypes to nematode antigen is likely due to the malaria parasite promoting Th1 cytokines in the environment where the antibody response is established [
45]. The biological consequences of the
Pcc driven IgG2a response to the nematode antigen and the less pronounced IgG1 response of
Nb mice to
Pcc antigens remain to be investigated. In
Trichuris muris infection, manipulation of the immune environment to a Th1 type setting, characterised by elevated IgG2a and IFNγ, was shown to enhance chronicity of this intestinal helminth [
62].
Pcc-induced IgG2a to nematode antigens may thus have real consequences in terms of disease outcome. Effects of nematode co-infection on the malaria parasite are also evident;
Pcc-Nb mice have reduced levels of malaria parasitemia in comparison to
Pcc mice [
53] and it is interesting to consider the possibility that cross-reactive IgG2a antibodies induced by the nematode infection may act in concert with the antigen-specific response to control malaria parasites. The potential for cross-reactive responses to have a functional role during infection raises the intriguing possibility that their production is a deliberate strategy of the host to combat diverse parasites [
63]. To fully understand the relative contribution of cross-reactive antibodies in parasite control would require passive antibody transfer experiments.
Although schistosome parasites are phylogenetically distinct from nematodes, helminth co-infection studies that investigate
Schistosoma mansoni provide evidence that cross-reactivity is relevant in other co-infection systems and can have a strong impact on disease severity. Naus et al [
43] report the induction of cross-reactive IgG3 antibodies that recognise both
Plasmodium falciparum and
S. mansoni antigens. Pierrot et al extended this study, identifying the
S. mansoni antigen (SmLRR) that is recognised by both malaria and
S. mansoni singly-infected hosts. Interestingly, as we observed in our
Pcc-
Nb model of co-infection, the two infections induce different antibody isotypes to antigen: cross-reactive malaria driven IgG3 and helminth driven IgG4 [
45]. In areas co-endemic for these two parasites, exposure to malaria and subsequent induction of the cross-reactive IgG3 response seems to increase the risk of developing hepatosplenomegaly in schistosome infected individuals [
44].
Authors' contributions
KFC participated in the design of the study, conducted the Pcc-Nb experiments and immunoassays, performed the statistical analysis and helped to draft the manuscript. TL conducted the Pcc-Ls experiments and immunoassays. JL provided recombinant MSP-119. ALG conceived of the study and participated in its design, was involved in all co-infection experiments and helped to draft the manuscript. JEA also conceived of the study, participated in its design and helped to draft the manuscript. All authors read and approved the manuscript.