The recognition of antigens on the surface of adult and L4 Necator americanus by human and hamster post‐infection sera

Summary The surface antigens of adult Necator americanus were recognized by post‐infection hamster sera and resolved at molecular weight 93000, 67000, 46000, 43000, 32000 and 25000. L4 larvae in contrast had one major surface antigen, resolving at 93000. These antigens were also recognized by a range of human sera, although on a differential basis. This suggests that the human sera tion. However, the results do indicate that the hamster model might be of immunological relevance to the human disease state, in that infected hamster recognized the full cuticular antigen spectrum of adult Necator. This, at least, gives the experimenter a convenient reference point from which to conduct further experiments incorporating parameters such as re‐infection, anthelmintic treatment and genetic variability to study the effect of these modifications on the serological response.


Introduction
The cuticle of nematodes is a dynamic structure, capable of participating in a variety of functions. It is biochemically active, maintaining the homeostasis of the organism (Lumsden 1975, Lee 1977, performs as an elasticated exoskeleton (Inglis 1983) and presumably represents a barrier against immune attack. It is apparent, however, that this barrier can be overcome and this has served to focus attention on the antigenic properties of the nematode cuticle (Philipp & Rumjanek 1984). Little attention has been paid to nematodes which establish as chronic infections of the gastrointestinal tract, and the human hookworm Necator americanus is a prime example. The present study was initiated to resolve this situation and to determine: (a) whether the immune system of hosts harbouring Necator adults is capable of recognizing epitopes expressed at the parasite's surface; and, (b) whether the laboratory model of hookworm disease, the hamster, can be considered relevant to the human situation with regard to the recognition of surface antigens.

PARASITOLOGY
The infective larvae of N. americanus were obtained from Dr G. Rajasekariah in 1983 and were from a strain maintained in laboratory hamsters for 69 generations in India. During the course of this work the parasite was passaged a further five times in Nottingham and larvae obtained from these infections have been described by Sen & Seth (1967) and Behnke, Paul & Rajasekariah (1985). Hamsters 2-3 days old were infected percutaneously with 70-1 50 L3 and the infectivity of the inocula used was determined by worm counts within 3 weeks of infection. L4 and adult worms were hand-picked from the intestines of infected hamsters 17 and 35 days following infection respectively.

ANALYSIS OF ANTIGENS
Worms were labelled with 12sI using Iodogen and antigens and immunoprecipitates analysed by SDS-PAGE as described previously (Pritchard et al. 1984). Briefly, radiolabelled parasites were homogenised in the presence of protease inhibitors and centrifuged for 30 min at 1 1 000 g (4°C). Supernates were routinely assayed for labelling efficiency and counts were always between 40-58% TCA precipitable. Antigens and immunoprecipitates were run on slab gels consisting of a 12% running gel and 3% stacking gel. Dried gels were autoradiographed using Kodak X-OMATS film at -80°C.

SOURCE OF SERA
Hamster sera were obtained as shown in Table I., Human sera were obtained from hookworm infected individuals in the Gambia (P. Hagan) and Calcutta (G. Schad). Normal sera were taken from non-infected controls.

HAMSTER SERA
The different batches of N americanus larvae used in the present work were infective, with 50-70% of the administered dose being recovered within 3 weeks of infection. In all four experiments faecal egg counts were monitored in selected individuals from 5 weeks postinfection when eggs first appeared in the faeces until approximately 20 and 30 weeks in female and male hamsters respectively, when eggs could no longer be detected.
Using immune sera from infected hamsters to immunoprecipitate Iodogen-labelled adult Necator (SDS-PAGE analysis), it was revealed that the parasite possessed at least six distinct antigens accessible to surface-labelling and that these resolved under reducing conditions at mol wt. approximately 93 000, 67000 (doublet), 46000 (doublet), 43000, 32000 and 25000 (Figure 1). The immune response was qualitatively the same along a time course following infection in that each of these polypeptides was recognized although the degree of recognition increased during the course of infection. This was reflected in the number of ct/min precipitated (Table 1) and, consequently, the intensity of the autoradiography. Sera taken from male and female hamsters recognised the same epitopes on the parasites' surface (Figure 1). It was also interesting to note that there appeared to be an antigen specific to adult female worms, resolving at mol.wt approximately 25000 (Figure I-arrowed) and that the response to female worms was more pronounced overall.
The recognition of adult surface antigens by post-infection hamster sera was confirmed using pooled sera taken 95,108 and 125 days after infection ( Figure   Necator americanus males (58% TCA pptbl. 11 1329 ct/min-10 p1 pptd) and females (53% TCA pptbl. 190 132 ct/min-10 pI pptd) were homogenized in 10 mM Tris, pH 8.0 containing the protease inhibitors TPCK (50 pglml), TLCK (25 pg/ml), PMSF (1 mM) and 1 % (w/v) sodium deoxycholate. Homogenates were cleared and 10 p1 samples of the supernates taken for determination of the incorporation of 1251 into proteinaceous material. Antigens and immunoprecipitates were then analysed by SDS-PAGE as shown in figure 1. The L4 antigen used in later studies was 40% TCA pptbl. 21 329 ct/min-20 pl pptd.  Table 1 for details). The profile of these immunoprecipitates is shown in each of these figures in lanes 1-13. N.A.HOM = total surface labelled material. larvae using these sera (lanes 3,5,7). However, the other major cuticular polypeptide (mol wt. 41 000) did not seem to be precipitated under the conditions used in this experiment (lanes I , 12) (see Discussion Section).

HUMAN SERA
A sample of serum taken from a hookworm positive patient (courtesy of Dr P. Hagan,   lanes 1, 2) sera.

Discussion
The longevity of infections with N . americanus in man remains a controversial subject with reports varying in their estimates from  1978, Ball & Bartlett 1969, Schad, Soulsby, Chowdhury & Gilles 1975, Ogilvie et al. 1978 and includes antibody responses against L3 and adult stages. In the present study, the full spectrum of adult cuticular antigens recognized by the infected hamster was also recognized by a range of human sera, adding support to the relevance of the model to the human situation. Therefore this infers that the laboratory strain of N. americanus, which has been maintained for 74 generations in hamsters, still retains major cuticular antigens recognized by humans infected with field strains of this parasite. However, the alteration or deletion of surface antigens with possible retention of conserved determinants remains a possibility. The experimental model also has the advantage of being under the control of the investigator, and despite the relatively short period of parasite patency encountered in the hamster model compared with man, the model has much to offer in terms of providing life cycle stages hitherto unavailable for research and an opportunity to analyse hostprotective responses.
The major adult cuticular antigens recognized by the immune system resolved at mol wt. 93000,67000,46000,43000,32000 and 25000, although the latter might be peculiar to female parasites. The differential recognition of surface antigens by sera from hookworm positive patients could reflect differences in the degree of longevity of infection and accompanying parasitological and clinical data will become increasingly important as these studies progress. Cross-reactivity with other parasites and the genetic heterogeneity of the host population are also parameters which will warrant close scrutiny.
To consider the antigens themselves in greater detail, it is relevant to note that a mol wt. antigen 67000 is also produced in measurable amounts by adult Necator during in uitro culture (Carr & Pritchard, unpublished), indicating that the cuticle of Necator could be involved in both antigen presentation and secretion in a manner similar to that described by Maizels, de Savigny & Ogilvie (1984). In this work, Toxocara canis larvae were shown to shed 25% of their surface antigens in 1 h of culture (an estimated 200 pg of ES protein/larva/day). Support for a cuticular contribution to ES comes from the demonstration that adult Necator sheds significant quantity of its surface antigen into the surrounding medium during (in uitro) culture (Pritchard et al. 1986).
Alternatively, ES antigens may be adsorbed onto the surface. Such a mechanism has been suggested to be operative for an excretory-secretory antigen of Trichinella spiralis (Silberstein & Despommier 1985) on the basis of reactivity of defined monoclonal reagents against ES, the cuticular surface and the lining of the gut.
It will also be interesting to determine whether epitopes resolving around mol.wt 32000 bear any resemblance to the major accumulated antigens of adult Necator encountered by Western blotting (30000-35 000 mol wt.-unpublished) and the proteolytic enzyme (37000) recently described in Ancylostoma caninurn by Hotez et al. (1985). Finally, the recognition of an antigen epitope at 93000 on L4 larvae by long-term infection sera paves the way for studies on antigenic homology between larval and adult stages. It is also worth noting that the failure of long term infection sera to precipitate the 41 000 surface antigen of L4 larvae is consistent with the observation that only short term (day 17) post-infection sera recognized a 42000 L4 stage specific ES product (Carr & Pritchard-unpublished). Monoclonal reagents should reveal any homology which might exist between ES products and cuticular epitopes on both L4 and adult stages.
Therefore, in conclusion, it is felt that the results described above indicate that the hamster model bears immunological relevance to the human situation, at least as far as adult surface antigens are concenred. However, a major requirement to complete these studies in the provision of human post-infection sera with accompanying parasitological and clinical data.