IgG subclass recognition of Loa loa antigens and their correlation with clinical status in individuals from Gabon

IgG subclass recognition of Loa loa antigens and their correlation

with clinical status in individuals from Gabon

J.P.AKUE1,2 M.HOMMEL 2 & E.DEVANEY 3

1 Centre International de Recherches Medicales de Franceville, BP 769, Franceville, Gabon2 University of Liverpool, Liverpool School of Tropical Medicine, Pembroke Place Liverpool L3 5QA, UK3 University of Glasgow, Department of Veterinary Parasitology Veterinary School, Bearsden Road, Glasgow G61 1QH, Scotland, UK

SUMMARY

In endemic areas forLoa loa,a significant percentage ofactively infected individuals have no circulating micro-filariae, an observation which implies the existence of astage-specific immune response. In an attempt to define theimmunological basis of the amicrofilaraemic state, thereactivity of antigens from adult, microfilariae and infectivelarvae of L. loa was examined by Western blotting withindividual serum samples from four clinically definedgroups (high microfilaraemic, low microfilaraemic, amicro-filaraemic and endemic controls) using IgG subclass-specific reagents and IgE. In the adult parasite, a complexof antigens at 28-31 kDa was exclusively recognized byIgG1 from amicrofilaraemic individuals and, to a lesserextent, by IgG1 from endemic controls. However, thiscomplex of antigens was recognized by IgG4 antibodies inserum samples from all individuals, including microfilar-aemics. A microfilarial antigen of 21 kDa was recognized byIgG1 antibodies present in serum from amicrofilaraemic,endemic control and low microfilaraemic individuals. Per-sons with high levels of microfilariae did not recognise thisantigen. In both the L3 and the microfilariae, a ladderantigen with increments of 15 kDa was the main target ofIgG4 antibodies in amicrofilaraemic and microfilaraemicindividuals. IgE antibodies recognized more antigens in themicrofilarial stage than in the adult of L3. These resultssuggest that immunological differences between clinicallydefined groups are associated with the recognition ofdifferent antigens or epitopes.

Keywords Loa loa, IgG subclasses, differential antigenrecognition

INTRODUCTION

The filarial nematodeLoa loa remains a significant publichealth problem in the West African forest block, where itrepresents the third cause for visits to rural hospitals(Boulestiex & Carme 1986). The infection is transmittedby the bite of aChrysopscarrying third stage larvae (L3)within the head and mouthparts. The L3 develop to adults inthe subcutaneous tissues and the female worm producesmicrofilariae (mf) which circulate in the bloodstream. Aninteresting feature of loiasis is that two thirds of individualsliving in endemic areas are amicrofilaraemic, despite beingcontinually exposed to infective bites (Van Hoegaerdenet al.1987). However, many amicrofilaraemic individuals areactively infected as demonstrated by the ocular passage ofan adult worm, or by positive serological tests (Akue,Hommel & Devaney 1996). These observations imply theexistence of a stage-specific immune response which mayclear mf from the circulation, in the absence of an effectiveimmune response against the adult parasite in the tissues.Previous studies in Gabon provided circumstantial evidencethat antibodies may have a role in the control of mf levels.For example, amicrofilaraemic individuals were shown tohave antibodies which recognized a 31 kDa antigen in adultL. loa (Egwanget al. 1988) and a 23 kDa antigen in mf(Pinder, Dupont & Egwang 1988), while serum from micro-filaraemic individuals did not recognize these antigens.However, both these studies were carried out by immuno-precipitation of125I labelled cuticular proteins using ProteinA, which binds all IgG subclasses in humans, except forIgG3 (Kronvall & Williams 1969). Thus, the analysis wasrestricted to surface/cuticular antigens and it was not pos-sible to correlate recognition of specific antigens with aparticular subclass response. In a further study, the recogni-tion of adult antigens by IgG subclasses in pooled loiasisserum was investigated (Egwanget al.1989). A correlationwas observed between resistance and high levels of IgG3antibodies.

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Correspondence: J.P. AkueReceived: 18 December 1997Accepted for publication: 6 April 1998

In lymphatic filariasis it has been proposed that thebalance between different IgG subclasses influences theclinical outcome of infection (Kurniawanet al. 1993) andour recent studies inL. loa infection have shown that thelevel of IgG subclasses varies according to clinical status(Akue, Hommel & Devaney 1997). Thus, IgG1 was elevatedin amicrofilaraemics and endemic controls, while levels ofIgG2, IgG3 and IgG4 were similar in all groups. Whetherthis variation in subclasses responses is due to the nature ofthe antigens recognized is not known. In this study, weattempted to identify adult, mf or L3 antigens that weredifferentially recognized by IgG subclasses in individualserum samples from different clinical groups. The recogni-tion of particular antigens by amicrofilaraemic individualsmay help to define the immunological mechanisms whichunderly the amicrofilaraemic state in loiasis.

MATERIALS AND METHODS

Study population

The study population has been described in detail elsewhere(Akue et al. 1996, 1997). The population consisted of 56adult volunteers from Ambinda, an endemic village inGabon; these individuals had been followed up over aperiod of five years and were grouped according to theirparasitological status as: amicrofilaraemic (n¼ 34), lowmicrofilaraemic (n¼ 9, mf levels<100 ml¹1), high micro-filaraemic (n¼ 6, mf levels> 100 ml¹1) and endemic con-trol (n¼ 7, no clinical or parasitological history ofinfection). In individuals grouped as ‘low microfilaraemic’,mf were not apparent upon direct examination of wet bloodsmears; the detection of mf required that blood samples beconcentrated. All amicrofilaraemic individuals were con-sidered to be actively infected on the basis of an occularpassage of an adult worm or elevated levels of antigen-specific IgG4 (Akueet al. 1997). The filarial nematode,Mansonella perstans, was also endemic in the village andwas equally distributed between the differentL. loa clinicalgroups (Akueet al. 1996). Caucasian sera were used asnegative controls.

Loa loa antigens

Adult L. loa were removed by an opthalmologist duringocular passage while mf were isolated from the blood ofheavily infected individuals. Mf were purified on Percollgradients as described previously (Van Hoegaerden &Ivanoff 1986). L3 were dissected from naturally infectedChrysops. Adult parasites and L3 stages were disrupted byhomogenisation in PBS while mf were sonicated in PBS andthen extracted by boiling in SDS sample cocktail containing

2% SDS and 100 mM dithiothreitol in Tris-HCl buffer, pH6.8. The protein content of each extract was estimated bytitration on Coomassie blue-stained mini-gels followed bydensitometric scanning.

SDS-PAGE and Western blotting

L. loa antigens were separated by SDS-PAGE under reduc-ing conditions on 12·5% acrylamide mini-gels (6×9 cm),with a discontinuous buffer system (Laemmli 1970). 100mgof protein was analysed in a continuous well. Separation wasoptimal over the Mr range 97–10 kDa, and only antigens inthis range were scored as differentially recognized. Follow-ing electrophoresis, proteins were transferred onto nitrocel-lulose paper (NCP) by electrotransfer in a buffer containing0·25 M Tris-HCl, 0·192 M Glycine, 20% Methanol pH 8.3(Towbin, Staehelin & Gordon 1979). The NCP was stainedwith Ponceau S (Sigma Chemical Co., St. Louis, MO, USA)to visualize the bands, cut into strips, then blocked for anhour in Tris-Buffered Saline, 0·05% Tween 20 (TBST) with3% BSA. Following three washes in TBST, each strip wasincubated overnight at 48C with serum from individualpatients diluted 1/200 or 1/20 (for IgE detection) in TBST1% BSA. In the initial experiments, strips were probed witha pool of serum that comprised 10ml from each individual inthe four separate groups. The strips were washed and thenincubated with mouse monoclonal antibodies against humanIgG subclasses (Oxoid, Unipath, Hampshire, UK) for 2 hat room temperature. The antibodies used were IgG1(HP6012) at 1/2000, IgG2 (HP6014) at 1/2000, IgG3(HP6050) at 1/1000 and IgG4 (HP6011) at 1/30000 dilutedin TBST 1% BSA. After washing as above, the NCPstrips were incubated for one hour with anti-mouse IgG(Fc-specific) conjugated to alkaline phosphatase, diluted1/1000 in TBST 1% BSA. For detection of IgE, stripswere incubated in 1/400 anti-human IgE (Sigma) in TBST1% BSA. This was followed by another washing step andincubation in the substrate solution containing 5 bromo-4-chloro-3-Indolyl (BCIP, 0·3 mg/ml) and Nitro Blue-Tetra-zolium (NBT, 0·15 mg/ml) in 1 M Tris-HCl, 500 mM MgCl2buffer, for visualization of bound antibodies. Blots werescored by visual inspection to identify particular compon-ents that were recognized by IgG subclasses from thedifferent clinical groups.

RESULTS

Identification of antigens in the adult stage recognizedby different IgG subclasses

In the initial experiments, adultL. loa antigen was probedwith a pool of serum from each of the four clinical groups,

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followed by the different subclass reagents (Figure 1). Thispreliminary analysis demonstrated that IgG1 antibodiesfrom amicrofilaraemics (Figure 1, G1, lane a) exclusivelyrecognized a complex of three antigens at 28–31 kDa.These antigens were also weakly recognized by IgG1 fromendemic controls (Figure 1, G1, lane b), but not by IgG1from low or high microfilaraemics (Figure 1, G1, lanes c andd, respectively). Recognition of this complex of antigenswas weak for IgG2 (Figure 1, G2), and IgG3 (Figure 1, G3).However, IgG4 antibodies from each of the four clinicallydefined groups recognized all the components of thecomplex (Figure 1, G4). A component at approximately40 kDa was also recognized by IgG1 and IgG3 fromamicrofilaraemic, low microfilaraemic and endemic controlindividuals.

In the subsequent analyses, serum samples from eachindividual patient were used to probe the blots and therecognition of individual antigens was recorded and plottedaccording to clinical status. This analysis allowed differ-ences in the pattern of antigen recognition to be comparedbetween the clinical groups. For ease of presentation, only asubset of individual sera are shown in the Figures.

Figure 2 illustrates a representative blot carried out withadult antigen and individual serum samples and developed

with the anti-IgG1 reagent. The majority of antigens pre-ferentially recognized by amicrofilaraemics were alsorecognized by a small portion of microfilariae carriers. Incontrast to the pattern seen with other antigens, the complexof antigens ranging in size from 28 to 31 kDa was almostexclusively recognized by IgG1 from amicrofilaraemicindividuals. The 31 kDa component was weakly recognizedby IgG1 from some endemic control individuals and somelow microfilaraemics, whereas the lower band of 28 kDawas recognized by amicrofilaraemics and endemic controlsonly. The recognition of these component bands by endemiccontrol and low microfilaraemic individuals was apparentonly when the blots were freshly developed. Blots withindividual serum samples and IgG2, IgG3 and IgG4 reagents(data not shown) generally confirmed the results obtainedwith pooled serum. As described with pooled serum,100% of individual serum samples tested contained IgG4antibodies which recognized the 28–31 kDa complex.

A range of additional antigens were differentially recog-nized by IgG1 antibodies from amicrofilaraemic andendemic control sera compared to microfilariae carriers;these included components at 56 kDa, 44 kDa, 42 kDa,40 kDa and 14 kDa. However, none of these antigens wasexclusively recognized by amicrofilaraemic subjects.

Identification of microfilarial antigens recognized byIgG subclasses

When mf antigens were analysed, the most striking differ-ence was the preferential recognition of a 21 kDa antigen byIgG1 of most amicrofilaraemic and some endemic controlindividuals. The 21 kDa antigen was weakly recognized byonly two low microfilaraemics, and not at all by highmicrofilaraemics as shown on the blot in Figure 3. Inaddition to the 21 kDa antigen, several other proteins at32 kDa, 40–42 kDa and 97 kDa were recognized morefrequently by amicrofilaraemics compared to microfilarae-mics (arrowed in Fig. 3). A doublet at 14 kDa was recog-nized by IgG1 from individuals in all parasitologicalcategories. Interestingly, a protein of 21 kDa was alsorecognized by IgG3 in 38% of amicrofilaraemics, but noneof the serum samples from high microfilaraemic individualscontained IgG3 antibodies reacting with this component(data not shown). In contrast to the pattern of recognitionobserved with other subclasses. IgG4 antibodies from aproportion of individuals in each of the clinical groups(except the endemic control group) recognized a prominentantigen, which appeared as a ‘ladder’ with increments of15 kDa (Figure 4). IgG2 from endemic control sera prefer-entially recognized a minimal number of proteins in mfextract, but the level of binding was very low, and the blotsdid not show a clear cut pattern (data not shown).

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Figure 1 Comparative binding pattern of IgG subclasses to adultL. loa antigens. Western blots of adultL. loa antigen were preparedas described in Materials and Methods. The NCP was cut into stripsand each strip probed with pooled serum from amicrofilaraemics(lanes a), endemic controls (lanes b), low microfilaraemics (lanes c)and high microfilaraemics (lanes d) at a 1/200 dilution. The blotswere developed with anti-human IgG1 (Panel G1), anti-human IgG2(Panel 2), anti-human IgG3 (Panel G3), and anti-human IgG4 (PanelG4), followed by anti-mouse IgG alkaline phosphatase conjugate. Theposition of the 28-31 kDa complex is marked with arrows. Molecularweight standards are indicated on the left.

Identification of infective larval antigens recognized byIgG subclasses

When homologous L3 were used as the source of antigen,the prominence of the IgG1 and IgG4 reactivity over IgG2and IgG3 was obvious, as shown by the number of antigens(approximately 25) recognized by IgG1 (Figure 5), andIgG4 (approximately 32) (Figure 6), and the intensity ofthe signal compared to IgG2 or IgG3 (data not shown). ForIgG1, L3 proteins preferentially recognized by amicrofilar-aemic and endemic control sera were also recognized bysome microfilaraemic individuals, though with a lowerfrequency. L3 antigens differentially recognized by IgG1from amicrofilaraemic serum were at 14 kDa, 22 kDa and76 kDa. As with mf antigen, the pattern of IgG4 reactivitywas dominated by the ‘ladder’ antigen in individuals fromthe amicrofilaraemic and low microfilaraemic groups, butnot in the high microfilaraemic or endemic control group(Figure 6).

Recognition ofL. loa antigens by IgE

Blots were also carried out with IgE specific reagents andantigen from all three life cycle stages. For all three antigen

preparations, most reactivity was observed with IgE anti-bodies from amicrofilaraemic individuals, despite the obser-vation made in a previous study (Akueet al. 1997), thatthere was no significant difference in levels of IgE anti-bodies in the different clinical groups. Adult antigen reactedpoorly with IgE as did L3, while most binding was observedto mf antigens (data not shown). However, no distinctivepattern was observed for IgE binding to mf antigensamongst the different clinical groups.

DISCUSSION

This study revealed a number of interesting findings withrespect to the recognition of particularL. loa antigens bydefined IgG subclasses. In the adult extract a complex ofantigens at 28–31 kDa was recognized by IgG1 antibodiesin amicrofilaraemics and endemic controls, suggesting thatthe recognition of these antigens by IgG1 may be correlatedwith the amicrofilaraemic state. Cuticular antigens of 29–31 kDa were previously identified in adultL. loaby labellingwith 125I and were also found to be recognized preferentiallyby total IgG from amicrofilaraemic and resistant individuals(Egwanget al. 1988). It has yet to be formally proved that



Figure 2 Identification of adultL. loa antigensby specific IgG1 antibodies. Western blots ofadultL. loa antigen were prepared as describedin Materials and Methods. The NCP strips wereprobed with individual human serum fromnegative control (C), high microfilaraemics(HMF), low microfilaraemics (LMF), endemiccontrols (EC) and amicrofilaraemics (AMF) at1/200 dilution. The blot was developed withanti-human IgG1 and anti-mouse IgG alkalinephosphatase conjugate. The position of thedifferentially recognized antigens are markedwith arrows; molecular weight standards areindicated on the left.

Figure 3 Identification ofL. loa microfilarialantigens by specific IgG1 antibodies. Westernblots ofL. loa mf antigens were prepared asdescribed in Materials and Methods. The NCPstrips were probed with individual humanserum from negative control (C), highmicrofilaraemics (HMF), low microfilaraemics(LMF), endemic controls (EC), andamicrofilaraemics (AMF) at 1/200 dilution.The blot was developed with anti-human IgG1and anti-mouse IgG alkaline phosphataseconjugate. The position of the differentiallyrecognized antigens are marked with an arrow;molecular weight standards are indicated onthe left.

the 125I labelled cuticular antigens and the complexdescribed in the present study are homologous. Furthermore,it remains unclear whether the 28–31 kDa complex ofantigens derives from processing of a single gene productor whether the components of the complex represent theproducts of different genes. Treatment of the125I labelledcomplex with Endoglycosidase H was shown to reduce theantigenicity of the complex (Egwanget al. 1988). Interest-ingly, IgG1 antibodies fromL. loa amicrofilaraemics alsorecognized a single component of 29 kDa from the adultworm of the heterologous filarial parasite,Brugia pahangi(data not shown). The antigen recognized inB. pahangiisthought to be gp29, the cuticular glutathione peroxidase(Cookson, Blaxter & Selkirk 1992). However, an associa-tion between the recognition of gp29 and resistance has notbeen reported in lymphatic filariasis (Maizelset al. 1987).Preliminary experiments using PCR primers designed fromthe sequence of theBrugia gp29, suggest that an homo-logous gene exists inL. loa (Akue 1995); attempts areunderway to clone and express the putativeLoa homologueso that the pattern of IgG subclass binding to a definedantigen can be studied.

A striking observation in the present study was that whilethe recognition of the 28–31 kDa complex was restricted toIgG1 antibodies from amicrofilaraemics and endemiccontrols, a complex of antigens of the same molecularweight was recognized by IgG4 antibodies from 100% of

individuals. Thus the same antigens appear to be differen-tially recognized by different IgG subclasses according toclinical status. Since the results of our experiments derivedfrom the same gels and antigen preparations, the differentialrecognition of the 28–31 kDa complex is likely to be a truephenomenon, rather than an artefact due to technical varia-tion. The parallel recognition of this complex by IgG1 andIgG4 is reminiscent of the results of Hussain & Ottesen(1986), who demonstrated that the same antigens wererecognized by IgG4 and IgE in lymphatic filarial infection.IgG4, which is particularly elevated in lymphatic filariasis(Kurniawanet al. 1993, Maizelset al. 1995), may ‘block’potentially protective or pathogenic responses. In loaisis,our previous studies have revealed no differences in IgG4levels between different clinical groups (Akueet al. 1997),with endemic controls having equivalent levels ofLoa-specific IgG4 as the other groups. Endemic controls arecontinuously exposed to L3, and, in the absence of a markerof current infection, may be an heterogenous group. Whilethe factors which drive expression of a particular subclassresponse inL. loa infection are not known, IgG4 appears tobe stimulated by stages other than the microfilariae(Akue, Egwang & Devaney 1994). The elevated levelsof Loa-specific IgG1 in amicrofilaraemic individuals mayindicate that this subclass is down-regulated in the presenceof microfilariae.

In O. volvulus infection, Steelet al. (1990) reported



Figure 4 Identification ofL. loa microfilarialantigens by specific IgG4 antibodies. Westernblot of L. loa mf antigens were prepared asdescribed in Materials and Methods. The stripswere probed with individual human serum fromnegative control (C), high microfilaraemics(HMF), low microfilaraemics (LMF), endemiccontrols (EC) and amicrofilaraemics (AMF) at1/200 dilution. The blot was developed withanti-human IgG4 and anti-mouse IgG alkalinephosphatase conjugate. Molecular weightstandards are indicated on the left.

Figure 5 Identification ofL. loa L3 antigens byspecific IgG1 antibodies. Western blots ofL. loaL3 antigens were prepared as described inMaterials and Methods. The NCP strips wereprobed with individual human serum fromnegative control (C), high microfilaraemics(HMF), low microfilaraemics (LMF), endemiccontrols (EC), and amicrofilaraemics (AMF) at1/200 dilution. The blot was developed withanti-human IgG1 and anti-mouse IgG alkalinephosphatase conjugate.

differential recognition of a defined molecule, paramyosin,by different IgG subclasses; they examined antibodyresponses to paramyosin, and showed that IgG3 responseswere correlated with the level of mf while IgG2 and IgEresponses appeared to be correlated with resistance. Theseobservations suggest that different components of the com-plex or different epitopes on the same molecule maystimulate different subclass responses and highlight thefact that recognition alone is not sufficient to define aprotective antigen.

In the mf stage of the life cycle, an antigen of 21 kDa wasdifferentially recognized by IgG1 antibodies from amicro-filaraemic and endemic control individuals. The same anti-gen was poorly recognized by low microfilaraemics and wasnot recognized at all by high microfilaraemics. A 23 kDasurface antigen was previously described in the mf ofL. loa,the recognition of which was correlated with the amicrofi-laraemic state (Pinderet al. 1988). As with the adult 28–31 kDa complex of antigens, additional studies are requiredto establish the possible relationship between the mf 23 kDasurface antigen and the 21 kDa antigen identified in thepresent study.

Western blots with IgG4 confirmed the prominence ofthis subclass over the other subclasses, as both the number ofantigens recognized and their intensity were greater com-pared to other subclasses. Interestingly, the predominantantigen recognized by IgG4 antibodies in mf and L3 stagesof L. loa was a ‘ladder’ antigen, so called because the15 kDa increments in size which result from processing ofa large precursor molecule (Ajuhet al. 1995). A similarantigen has been identified in other parasitic nematodes(Culpepperet al.1992, Pooleet al.1992, Spenceet al.1993,Tweedieet al.1993), and the genes which encode the ladderantigen inBrugia (Tweedieet al. 1993) and inLoa (Ajuhet al.1995) share a high degree of sequence homology withABA-1, the Ascaris allergen (McGibbonet al. 1990,Christieet al.1990). In Brugian filariasis the ladder antigenis known to be the target of specific IgE responses (Paxtonet al. 1993), but in L. loa the ladder antigen was not

recognized by IgE antibodies. Serum samples from someindividuals in the low microfilaraemic, amicrofilaraemic,and endemic control groups, but not the high microfilar-aemic group, contained specific IgG1 which bound to theladder antigen. The antigenicity of this molecule inL. loainfection was suggested by experiments in which aL. loagenomic expression library was screened with pooled ami-crofilaraemic serum. This resulted in the isolation of a clonewhich was recognized by amicrofilaraemic serum only(Egwang, Pinder & Akue 1990), and which was subse-quently shown to encode the ladder antigen (Ajuhet al.1995). These observations indicate that the role of the ladderantigen and its relationship to immune status inLoainfection have yet be fully clarified.

Although a number of L3 antigens were differentiallyrecognized by serum from amicrofilaraemic individuals, nosingle antigen was exclusively recognized by 100% ofamicrofilaraemic sera. It was therefore not possible tocorrelate the recognition of a particular L3 antigen by adefined subclass with resistance, despite the fact that ourprevious studies demonstrated an inverse correlationbetween levels of IgG1 antibodies to L3 antigens and mflevels (Akueet al.1997). These observations emphasize thefact that the mechanisms of resistance which result in theamicrofilaraemic state in loiasis are incompletely under-stood and likely to be complex. However, the results of thisstudy have identified a number of antigens in different lifecycle stages, the recognition of which appear to be corre-lated with the amicrofilaraemic state. Attempts are nowunderway to clone and further characterise some of thesemolecules, in the hope that the analysis of subclass-specificresponses to defined antigens may shed further light uponthe immune response in loiasis.

ACKNOWLEDGEMENTS

CIRMF is supported by the state of Gabon, Elf Gabon, andMinistere de la cooperation francaise. E.D. is a WellcomeTrust university lecturer.



Figure 6 Identification ofL. loa L3 antigens byspecific IgG4 antibodies. Western blot ofL. loaL3 antigens were prepared as described inMaterials and Methods. The NCP strips wereprobed with individual human serum fromnegative control (C), high microfilaraemics(HMF), low microfilaraemics (LMF), endemiccontrol (EC) and amicrofilaraemics (AMF) at1/200 dilution. The blot was developed withanti-human IgG4 and anti-mouse IgG alkalinephosphatase conjugate. Molecular weightstandards are indicated on the left.

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IgG subclass recognition of Loa loa antigens and their correlation with clinical status in individuals from Gabon