Vaccination-induced protection of lambs against the parasitic nematode Haemonchus contortus correlates with high IgG antibody responses to the LDNF glycan antigen

Lonneke Vervelde1,2, Nicole Bakker2, Frans N.J. Kooyman2, Albert W.C.A. Cornelissen2, Christine M.C. Bank3, A. Kwame Nyame4, Richard D. Cummings4 and Irma van Die3

2 Utrecht University, Department of Infectious Diseases and Immunology, Division of Parasitology and Tropical Veterinary Medicine, PO Box 80.165, 3508 TD Utrecht, The Netherlands; 3 VU University Medical Center, Department of Molecular Cell Biology and Immunology, Glycoimmunology Group, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands; and 4 University of Oklahoma Health Sciences Center, Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, Oklahoma City, OK 73104

Received on June 20, 2003; revised on July 14, 2003; accepted on July 17, 2003


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Lambs respond to vaccination against bacteria and viruses but have a poor immunological response to nematodes. Here we report that they are protected against the parasitic nematode Haemonchus contortus after vaccination with excretory/secretory (ES) glycoproteins using Alhydrogel as an adjuvant. Lambs immunized with ES in Alhydrogel and challenged with 300 L3 larvae/kg body weight had a reduction in cumulative egg output of 89% and an increased percentage protection of 54% compared with the adjuvant control group. Compared to the adjuvant dimethyl dioctadecyl ammonium bromide, Alhydrogel induced earlier onset and significantly higher ES- specific IgG, IgA, and IgE antibody responses. In all vaccinated groups a substantial proportion of the antibody response was directed against glycan epitopes, irrespective of the adjuvant used. In lambs vaccinated with ES in Alhydrogel but not in any other group a significant increase was found in antibody levels against the GalNAcß1,4 (Fuc{alpha}1,3)GlcNAc (fucosylated LacdiNAc, LDNF) antigen, a carbohydrate antigen that is also involved in the host defense against the human parasite Schistosoma mansoni. In lambs the LDNF-specific response increased from the first immunization onward and was significantly higher in protected lambs. In addition, an isotype switch from LDNF-specific IgM to IgG was induced that correlated with protection. These data demonstrate that hyporesponsiveness of lambs to H. contortus can be overcome by vaccination with ES glycoproteins in a strong T-helper 2 type response–inducing aluminum adjuvant. This combination generated high and specific antiglycan antibody responses that may contribute to the vaccination-induced protection.

Key words: carbohydrate-specific antibody response / excretory-secretory products / fucosylated LacdiNAc / Haemonchus / N-glycans


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Haemonchus contortus is a parasitic nematode in sheep and goats that causes significant economic losses worldwide due to its blood-feeding behavior. Vaccination may be a useful approach to control gastrointestinal nematode infections in ruminants, because alternatives are lacking and antihelminthic drug resistance in H. contortus is widespread (Van Wijk et al., 1997Go). In previous studies we have shown that excretory/secretory (ES) glycoproteins of adult H. contortus can induce protection against challenge infection. In all these studies dimethyl dioctadecyl ammonium bromide (DDA) was used as an adjuvant to induce both humoral and cellular responses in adult sheep and lambs, but protection was only induced in adult sheep (Schallig and Van Leeuwen, 1997Go; Vervelde et al., 2001Go).

Although lambs respond successfully to immunization against infection with bacteria and viruses, they have a poor immunological response to nematodes, such as H. contortus. The mechanisms of this hyporesponsiveness are not fully understood but could include feedback inhibition of maternally derived antibodies and colostral transfer of soluble antigen or soluble suppressor factors (Soulsby, 1985Go). Moreover, lower numbers of CD4+ and CD8+ cells in blood and skin and lower levels of specific antibodies have been described in lambs (Colditz et al., 1996Go; Kambara and McFarlane, 1996Go; Rudd et al., 2001Go; Vervelde et al., 2001Go).

Glycosylation may contribute significantly to the overall immunogenicity of a glycoprotein, especially when the glycan structure is foreign to the host. In human and murine schistosomiasis, glycoconjugates that abundantly occur on different stages of the parasite are a major focus of the host immune response (Cummings and Nyame, 1999Go; Nyame et al., 2000Go; Eberl et al., 2001Go; Van Remoortere et al., 2001Go, 2003Go; Van der Kleij et al., 2002Go; Kantelhardt et al., 2002Go). In particular, fucosylated glycoconjugates are prominently involved in the host's humoral and cellular immune responses to schistosomes (Eberl et al., 2001Go; Nyame et al., 2000Go; Van Remoortere et al., 2001Go; Van der Kleij et al., 2002Go; Kantelhardt et al., 2002Go). We hypothesized that fucosylated glycan antigens may be similarly important in the immune response against H. contortus and that an impaired response of lambs to nematode glycans may play a role in the hyporesponsiveness of lambs. In H. contortus, several core-fucosylated N-glycans have been identified that may be targets of the host immune response (Haslam et al., 1996Go; Van Die et al., 1999Go). Interestingly, the detection of high levels of an {alpha}1,3-fucosyltransferase activity (DeBose-Boyd et al., 1998Go), and the absence of Lewis x structures in H. contortus (Nyame et al., 1998Go) predicts the possible presence of the glycan antigen GalNAcß1,4(Fuc{alpha}1,3)GlcNAc (fucosylated LacdiNAc, LDNF), an antigen involved in the host defense against Schistosoma masoni (Eberl et al., 2001Go; Nyame et al., 2000Go).

Advances in parasite immunology have strongly indicated a crucial role for T-helper 2 type effector mechanisms in the rejection response of helminth parasites. In a murine schistosomiasis model, glycans have been shown to contribute to the induction of T-helper 2 type responses (Okano et al., 1999Go, 2001Go). In the design of effective, nonreplicating vaccines, immunological adjuvants serve as critical components that instruct and control the selective induction of the appropriate type of antigen-specific immune response. The effects of aluminum adjuvants are partly elucidated and show a general ability to stimulate a T-helper 2 type response (Lindblad, 1995Go). In the present experiment we compared the effect of two adjuvants, DDA and Alhydrogel, on the level of protection and the induction of ES-specific antibodies in the periphery and locally in the abomasum in lambs. In particular, we focused on glycan-specific antibody responses to better understand the role of parasite-specific glycans in relation to protective immunity and hyporesponsiveness in lambs. Because several of the glycan antigens on H. contortus glycoproteins are also found in other helminth parasites, such as the human parasite S. mansoni, the results of these studies are of general interest in the study of helminth infections.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Vaccination-induced protection is dependent on the adjuvant
To determine the efficacy of a vaccine based on ES proteins given with either Alhydrogel or DDA, all experimental groups were immunized three times with this subunit vaccine and subsequently challenged with H. contortus L3 larvae (see Table I). At different time points, egg output and worm burden were determined. Shedding of eggs was detected from day 18 postchallenge onward except in group 4, in which eggs were first detected at day 21 postchallenge. The mean cumulative egg counts in the feces are given in Figure 1A. Lambs immunized with ES in Alhydrogel (group 4) had significantly lower egg output than both the adjuvant control group (group 5; P < 0.05) and the lambs from worm-free ewes immunized with ES in DDA (group 2; P < 0.01). Lambs born from worm-free ewes (group 2) shed 18% more eggs than the control group. The percentage reduction in cumulative egg output for lambs immunized with ES in DDA (group 3) or in Alhydrogel (group 4) was 42% and 89%, respectively. The worm burdens are expressed as mean percentage protection and given in Figure 1B. The percentage protection is calculated to correct for the differences in challenge dose. The protected lambs (group 4) had significantly fewer worms than unprotected lambs of group 2 (P < 0.05). Early L4, L4, and L5 larvae were not detected, and there was no difference in the number of female or male worms recovered.


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Table I. Experimental groups

 


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Fig. 1. (A) Mean cumulative number of eggs per g feces (epg day 56–88). (B) Mean number of abomasal worms at day of necropsy given as percentage protection (1 – individual worm count/challenge dose x 100%). In both A and B comparisons are made between the lambs of group 2–5 to investigate an effect of the adjuvants tested. Data shown represent means and SEM (error bars) with 5 animals per group, except for group 5 which consisted of 3 animals. Similar superscripts indicate significant differences between these groups, at a P < 0.01 and b P < 0.05. Group 2 (*) lambs are offspring of worm-free ewes of group 1, groups 3–5 lambs are offspring of conventional ewes.

 
Protected animals show high levels of ES-specific antibodies
To determine the efficacy of the different adjuvants to induce a humoral response, the ES-specific antibody responses were measured during the course of the experiment (90 days). The ES-specific IgG responses are depicted in Figure 2A. Soon after the second immunization, the lambs immunized with ES in Alhydrogel showed a significant increase in IgG compared to lambs immunized with ES in DDA (P < 0.05). Within 1 week after the third immunization onward (day 49), all ES-immunized groups showed an increase in ES-specific IgG and the titer was significantly higher in the lambs immunized with ES in Alhydrogel compared to the other lambs (P < 0.01). The difference between the groups that received ES in DDA was not significant independent of their age or the worm status of their mothers.



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Fig. 2. ES-specific antibody levels in serum of the IgG (A), IgA (B), and IgE (C) isotype during the course of the experiment. Total IgE levels in serum are shown in D. All antibody levels are expressed as a percentage of the absorbance of the reference positive serum (% positive) that was used in all ELISAs and in previous studies. The animals were immunized at day 0, 21, and 42 (arrowhead) and challenged 2 weeks after the last immunization (arrow). Data represent means and SEM (error bars) with 5 animals per group, except group 5, which consisted of 3 animals. Asterisks denote significant differences between protected lambs (group 4) and unprotected lambs (group 2 and 3), *P < 0.05, ***P < 0.01.

 
As shown in Figure 2B, a small but significant increase in ES-specific IgA (P < 0.05) was found in lambs immunized with ES in Alhydrogel compared with all other lambs after the second and third immunization. ES-specific IgA increased in all groups after challenge infection. Significant differences between protected (group 4) and unprotected lambs (groups 2 and 3) were found from day 74 onward (P < 0.05).

Serum levels of ES-specific IgE antibodies are depicted in Figure 2C. One of the ewes in group 1 showed a high ES-specific IgE titer from day 0 onward. The course of her IgE response was similar to that of the other ewes in group 1 but always remained higher. The ES-specific IgE of lambs immunized with ES in Alhydrogel increased after the second immunization and was significantly higher than the other ES-immunized lambs from day 74 onward (P < 0.01).

The course of the total IgE levels (Figure 2D) was similar to that of ES-specific IgE. Total IgE levels were significantly higher in the older ewes at the start of the experiment up to day 28 (P < 0.01). Thereafter the protected lambs (group 4) had an increase in total IgE due to immunizations that was significantly higher than the total IgE levels of the unprotected lambs (group 2 and 3; P<0.05) and similar to the levels observed in ewes.

Vaccinated animals show glycan-specific antibody responses
To determine whether part of the Haemonchus ES-specific immune response observed in the vaccinated animals is directed against glycan epitopes, we investigated the binding of ES-specific antibodies to periodate-treated ES. The lowest concentration of periodate that effectively destroys the glycan epitopes of ES was determined by testing whether biotinylated lectins recognize ES antigens before and after treatment of ES with different concentrations of periodate (5–20 mM). Treatment of ES with 5 mM periodate was sufficient to abrogate nearly all binding of concanavalin A, wheat germ agglutinin, and lentil agglutinin (data not shown).

Destroying periodate-sensitive carbohydrate structures on ES resulted in a marked decrease in the binding of antibodies of all isotypes of pooled sera from day 49 and day 70 at which time the peak of ES-specific responses were found (Figure 3). Especially in the protected animals, that is, lambs of group 4 and adult sheep of group 1, the level of IgG and IgA antibodies that recognize ES epitopes after treatment with 5 mM periodate was dramatically decreased. ES-specific IgM and IgE showed a moderate decrease in the binding to periodate-treated ES. For IgM, this decrease was proportionally larger in the lambs compared to adult sheep, whereas for IgE the most prominent decrease was observed in the protected adult sheep. These data indicate that a substantial part of all serum antibodies recognize periodate-sensitive carbohydrate epitopes on ES. For all Ig classes we found that the use of higher serum concentrations resulted in increased OD, showing that responses to protein epitopes were present but generally much lower than the antiglycan responses.



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Fig. 3. ES-specific serum Ig levels before and after disruption of the periodate-sensitive glycan epitopes on ES. Coated ES was treated with 0 or 5 mM periodate and binding of Igs in pooled sera of day 49 and 70 was investigated by ELISA in duplicate for IgG (A), IgM (B), IgA (C), and IgE (D). Data shown represent means and SEM (error bars) of adult sheep of group 1 (closed bars), lambs of group 2 (open bars), lambs of group 3 (vertical hatched bars), lambs of group 4 (gray bars), and lambs of the adjuvant control group 5 (horizontal hatched bars).

 
Protected lambs show high levels of antibodies against the LDNF glycan epitope
Previous studies suggest that, similar to several other parasites, H. contortus may express LDNF epitope (DeBose-Boyd et al., 1998Go). Using a monoclonal antibody that recognizes the LDNF epitope (Nyame et al., 2000Go), the expression of this glycan epitope was determined by western blotting (Figure 4). Proteins in extracts of all three stages reacted with the anti-LDNF antibody, indicating that the LDNF antigen is present.



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Fig. 4. Western blot analysis of extracts of H. contortus for the presence of glycoproteins carrying LDNF. Glycoproteins from extracts of ES, adult worms, and infectious larvae (L3) were separated by SDS–PAGE under reducing conditions. The presence of glycoproteins bearing the LDNF glycan epitope was identified by immunoblotting using monoclonal antibody SmLDNF1 as described in Materials and methods.

 
Because of the abundance of the LDNF antigen on ES, we subsequently studied whether the animals responded specifically to this glycan antigen. In adult sheep we did not find a response after the immunizations or after challenge. In contrast, protected lambs (group 4) showed significantly higher levels of LDNF-specific IgM (Figure 5A; P < 0.01) and IgG (Figure 5B; P < 0.05 to P < 0.01) than unprotected lambs (groups 2 and 3). Interestingly, in the protected lambs an Ig class-switch from LDNF-specific IgM to IgG was induced by the immunizations, indicating that T cell help has been involved in the response against the LDNF antigen. This was not observed in protected adult sheep.



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Fig. 5. LDNF-antigen specific antibody levels in serum. The animals were immunized at day 0, 21, and 42 (arrowheads) and challenged 2 weeks after the last immunization (arrow), and binding of IgM (A) and IgG (B) antibodies to coated LDNF antigen was determined by ELISA at different time points. LDNF-specific IgM (dotted line) and IgG (solid line) levels of lambs immunized with ES in Alhydrogel (group 4) are depicted in C. Data represent means and SEM (error bars) with 5 animals per group, except group 5, which consisted of 3 animals. Asterisks denote significant differences between protected lambs (group 4) and unprotected lambs (group 2 and 3), *P < 0.05, **P < 0.025, and ***P < 0.01.

 
Correlations between serum antibody levels and protection against H. contortus
For the rational development of a vaccine we need to know which immune responses lead to protection and subsequently characterize the glycoproteins involved in these protective immune responses. To determine whether ES-specific antibody levels could predict the induction of protection, correlations between parasitological parameters and antibody levels were calculated (Table II). Comparisons were made between the lambs of groups 2–5 to be able to discover an effect of the adjuvant without having an age-related effect. As parasitological parameters, the number of eggs secreted during the course of the experiment and the percentage protection were used. The antibody levels at day 56 are used to determine whether the antibody responses induced by the immunizations only could predict whether the animals were protected against subsequent challenge infection. Correlations at later time points, at the peak of the antibody response after challenge infection (day 67), indicated an effect of the responses to the immunizations in combination with the booster responses induced by the worms of the challenge infection. The results in Table II demonstrate that the high levels of ES-specific IgG and LDNF-specific IgG induced by the immunizations predicted reduced egg excretion and worm burden after the challenge infection. Further experiments are needed to investigate whether ES-specific IgG and in particular LDNF-specific IgG can transfer protection against H. contortus.


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Table II. Putative contribution of specific antibody responses to protection after vaccination with ES glycoproteins of H. contortus

 
Local antibody responses are induced in protected animals
To determine whether the peripheral immunizations with ES in Alhydrogel or DDA also had an effect on the local responses in the abomasum, we measured the ES-specific mucus Igs. The mucus ES-specific IgA titers are shown in Figure 6. Lambs immunized with ES in Alhydrogel had significantly (P < 0.01) higher levels of mucus ES-specific IgA than lambs from worm-free ewes immunized with ES in DDA. Mucus ES-specific IgE and IgG levels were low (data not shown). Histological examination of the scraped abomasal tissue showed that the lamina propria was not affected and only mucus was isolated. Moreover, there was no correlation between ES-specific mucosal IgA and peripheral IgA, indicating local production of Igs and not leakage from the lamina propria.



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Fig. 6. ES-specific IgA in mucus of the abomasa. Igs in mucus were collected as described in Material and methods. Binding of antibodies to coated ES was determined by ELISA. Data shown represent means and SEM (error bars) with 5 animals per group, except group 5, which consisted of 3 animals. Comparisons are made between the lambs of group 2–5 to investigate an effect of the adjuvants tested. Similar superscripts indicate significant differences between these groups, a P < 0.01, b P < 0.05.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In this study we succeeded in overcoming the low, nonprotective responses of lambs after vaccination with ES by using the strong T-helper 2 type response-inducing aluminum adjuvant Alhydrogel. We investigated which underlying immune responses induced protection and whether the worm status of the ewes during gestation, that is, conventional or worm-free, would affect the peptide and glycan-specific immune responses after vaccination. Lambs immunized with ES in Alhydrogel shed significantly less eggs than lambs immunized with ES in DDA. The worm status of the ewes was of influence on the egg output as lambs from worm-free ewes shed more eggs than the lambs from conventional ewes immunized similarly. The protected lambs and adults had a significantly lower worm burden, which was in line with the reduction in egg output.

In vaccination experiments, the choice of adjuvant is often crucial for mounting protection. Previous studies have shown, however, that neither the immune response needed for protection nor the effectiveness of a particular adjuvant is easily predictable (Jacobs et al., 1999Go; Watson et al., 1994Go). Vaccination of lambs with ES in Alhydrogel instead of DDA has a significant positive effect on the intensity and time of onset of the ES-specific responses. In fact, the DDA-immunized groups hardly showed any responses before challenge infection, which was found previously (Vervelde et al., 2001Go). The ES-specific IgG response of the protected (Alhydrogel-immunized) lambs was significantly higher than that of the protected (DDA immunized) adult sheep, whereas the ES-specific IgA and IgE responses were more or less similar. After challenge, the IgG levels only remained high in lambs immunized with ES in Alhydrogel, indicating that Alhydrogel also has an effect on the duration of the humoral response, which is of importance for the induction of long-term memory. Besides the higher ES-specific serum antibody levels, we also found higher ES-specific mucus IgA levels in protected lambs and adult sheep, indicating that although the immunizations were given subcutaneously, a local mucosal response was induced. The differences found in the number of eggs per gram feces between lambs of conventional or worm-free ewes after immunization with ES in DDA were not reflected in their ES-specific serum Igs.

Our findings suggest that through vaccination a particular repertoire of ES-specific antiglycan and peptide antibody responses can be induced that results in protection. Both the level of protection and the induced ES-specific humoral responses depend on the age of the animals and the adjuvant used. In particular the glycan-specific responses were high and correlated with protection. This demonstrates that glycans on ES proteins of H. contortus are potent immunogens. H. contortus expresses an {alpha}1,3-fucosyltransferase capable of synthesis of the fucosylated LacdiNAc glycan epitope (DeBose-Boyd et al., 1998Go). This LDNF antigen is prominently involved in the host immune responses against schistosome infections (Eberl et al., 2001Go; Nyame et al., 2000Go). S. mansoni secretes glycoproteins expressing high amounts of LDNF antigen in its host (Nyame et al., 2002Go). Using western blotting, we showed that the LDNF antigen is also abundantly found on secreted glycoproteins (ES) of H. contortus, which might contribute to the high immunogenicity of ES products.

It would be of interest to identify the proteins carrying the LDNF epitopes by a proteomics approach using 2D gels (Yatsuda et al., 2003Go). We show here that in lambs immunized with ES in Alhydrogel, a significant increase was found in LDNF-specific antibody titers. Remarkably, an isotype switch from IgM to IgG was found in the protected lambs, and the induction of LDNF-specific IgG titers correlated with protection. In contrast, and for reasons unknown, in adult sheep hardly any response to this glycan antigen was found, although high antibody responses were found against other glycan epitopes. These data indicate that specific glycan antigens on ES proteins, such as LDNF, may play an important role in the defense against H. contortus. It may be possible that young lambs, unless they are stimulated with a strong T-helper 2 type response-inducing adjuvant, are unable to generate the antiglycan humoral immune responses needed to succesfully expel the nematodes.

Our results are consistent with other studies that have indicated an important role for glycans in the host defense against parasitic helminths. Protective immunity to H. contortus has been induced in goats using immunoaffinity- isolated antigens that share a phylogenetically conserved carbohydrate epitope on gut and secreted antigens (Jasmer and McGuire, 1991Go; Jasmer et al., 1993Go), but no details of the antigenic structure are known. In Trichinella spiralis infections, rapid expulsion of the nematode in rat pups resulted from a direct interaction of specific antibody with tyvelose-containing glycoproteins on larval surfaces and in ES antigens (Ellis et al., 1994Go). Moreover, using an in vitro invasion assay, it was demonstrated that antibodies against tyvelose can protect epithelia without the assistance of inflammatory cells, soluble cofactors, or mucus (McVay et al., 1998Go).

The production of IgG to glycan epitopes, as found in this article, implies major histocompatibility complex class II restriction and T-cell help. Over the past decade more evidence has been obtained that glycosylated peptides can participate in the cellular immune response and that the contribution of the carbohydrate to T-cell recognition is greatly differentiated. Both direct recognition of carbohydrates by the T-cell receptor and recognition of a specific conformation of the peptide conferred by the presence of a glycan without specific interaction between T-cell receptor and carbohydrate have been found (reviewed by Lisowska, 2002Go; Rudd et al., 2001Go). Our data show that glycans on ES are very immunogenic and induce a T-cell-dependent response, which is modulated by the adjuvant used concurrently. Recently, we demonstrated that dendritic cells can bind and internalize Lewis x and LDNF antigens through interaction of the pathogen receptor dendritic cell-specific ICAM-3-grabbing nonintegrin (Appelmelk et al., 2003Go; Van Die et al., 2003). This may result in antigen presentation and explain the specific humoral immune responses against these glycan antigens found in schistosome and H. contortus–infected hosts. Although the differences between adult sheep and lambs are not completely understood, our data indicate that glycan-specific immune responses contribute to protection rather than form a smoke screen for the immune system, as suggested for schistosomes by Eberl et al. (2001)Go.

There is a clear need to expand our knowledge of the diversity of the antibody responses induced after vaccination with native proteins because successful, reproducible vaccinations of sheep against H. contortus or other nematode parasites with recombinant proteins have failed so far. In contrast, vaccinations with native proteins have been successful for decades. Our results suggest that recognition of the importance of parasite-specific glycan antigens in the host immune response may greatly enhance the development of future vaccines.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Parasites
A benzimidazole-sensitive strain of H. contortus, originally obtained from the Moredun Research Institute (Penicuik, U.K.) was used. Challenge larvae were stored less than 3 months at 10°C in water. ES glycoproteins for immunizations were collected by keeping adult H. contortus for 24 h in RPMI 1640 as described previously (Schallig et al., 1994Go). Culture media were concentrated by lyophilization, dissolved in phosphate buffered saline (PBS), and desalted using PD-10 columns (Pharmacia, Uppsala, Sweden). Protein concentration was determined with the Bradford assay. The concentrated samples were stored at -80°C.

Experimental design
All Zwart Bles sheep were reared and housed indoors under worm-free conditions and fed pellets and silage. Lambs were weaned at an age of 10 weeks. In Table I the experimental groups are depicted. Group 1 with adult sheep was included as a positive control group (Schallig et al., 1997Go; Vervelde et al., 2001Go). All sheep were immunized subcutaneously three times at 3-week intervals. The ES antigens (75 µg/sheep/immunization) were mixed in either DDA (2.5 mg in PBS) or Alhydrogel (0.5 mg in physiological salt; Superfos Biosector, Denmark) with a final volume of 1 ml per immunization. Two weeks after the last immunization, all sheep were challenged intraruminally with 300 L3 larvae/kg body weight, resulting in a dose of around 10,000 L3 for female lambs, 12,000 for male lambs, and 21,000 L3 for adult ewes.

Fecal samples were collected before the start of the experiment, before the challenge to check their worm-free status, and twice a week from day 14 postchallenge onward. Blood samples were collected twice a week. Sheep were killed at day 33 postchallenge in a commercial slaughterhouse. The abomasum was opened immediately, and the contents were collected. Fecal egg counts were made using the McMaster technique and expressed as eggs per g feces. Worm counts were performed according to Eysker and Kooyman (1993)Go. Worm burden is expressed as percentage protection to correct for the differences in challenge dose: 1-(individual worm count/individual challenge dose) x 100. All procedures with the animals were performed under the supervision of the University of Utrecht Council for Experiments on Animals, according to Dutch legislation.

Peripheral ES-specific antibody levels
Serum samples were collected and stored at -20°C until the assays were performed. Collection and preparation of ES antigen was as described previously (Schallig et al., 1994Go) with the modification that we used 10% trichloroacetic acid in the medium for precipitation of ES and 8 M urea for dissolving the ES pellet to concentrate and desalt the isolated ES material.

Microtiter plates (Greiner, Germany) were coated overnight at room temperature with 2 µg/ml ES in 0.05 M carbonate/bicarbonate buffer pH 9.6. After washing, plates were blocked with PBS with 0.1% gelatin (Serva, Heidelberg, Germany) and 0.05% Tween-20 for 1 h at 37°C. Plates were stored at -20°C until use. For all isotypes, incubations were done for 1 h at 37°C and 100 µl per well; sera were analyzed in duplicate. Before use in the IgE enzyme-linked immunosorbent assays (ELISAs), all sera were treated for 1 h at 56°C. Standard positive and negative controls (Vervelde et al., 2001Go, 2002Go) were included on each plate. All dilutions were made in PBS with 0.1% gelatin and 0.05% Tween-20. The results were expressed as a percentage of the absorbance of the reference positive serum (% positive serum), which was used in all ELISAs and used in previous studies (Vervelde et al., 2001Go, 2002Go).

For the ES-specific IgG ELISA, the sera were diluted 1/200, for ES-specific IgA 1/100, and for ES-specific IgE 1/5. Plates were washed and incubated with mouse anti-sheep IgG or mouse anti-sheep IgA (Serotec, Kidlington, United Kingdom) or mouse anti-sheep IgE (IE7; Kooyman et al., 1997Go) diluted 1/400. The plates were washed and incubated with alkaline phosphatase conjugated goat anti-mouse (Dako, Denmark) diluted 1/3000 and subsequently developed with p-nitrophenyl phosphate disodium salt (PNPP, Pierce, Rockford, IL) for 30 min at room temperature followed by an overnight incubation at 4°C. The absorbance was measured at 405 nm using a Ceres UV900C ELISA reader.

To determine total IgE levels the plates were coated overnight at room temperature with 6 µg/ml IE7 in 0.05 M carbonate/bicarbonate buffer (pH 9.6). Sera were diluted 1/50, followed by incubation of 1/200 diluted polyclonal anti-sheep IgE (Kooyman et al., 1997Go). Alkaline phosphatase–conjugated goat anti-rabbit (Dako) was added at 1/3000 dilution, and plates were developed with PNPP.

Peripheral glycan-specific antibody levels
To determine the contribution of glycan-specific antibodies in the response against ES, periodate-sensitive carbohydrate epitopes were damaged. Microtiter plates were coated overnight with ES as described, washed three times with aquadest with 0.05% Tween-20 followed by 2 washes with 0.1 M sodium acetate (pH 4.5) and incubated for 1 h at 37°C with 5, 10, or 20 mM sodium m-periodate (Sigma, St. Louis, MO) dissolved in 0.1 M sodium acetate or with sodium acetate only. After two washes with sodium acetate and one with PBS, all plates were treated with 50 mM sodium borohydride (Sigma) in PBS for 30 min at room temperature. The plates were washed five times with 10 mM Tris–HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween-20 and blocked with 5% Elk (Campina Melkunie, Netherlands) in PBS for 1 h at 37°C. For this assay, pooled sera from day 49 and 70 were used, at which time the peak of ES-specific responses were found after the third immunization and after challenge. Sera were serially diluted from 1/200 to 1/12.5 for IgG, from 1/200 to 1/25 for IgM, from 1/100 to 1/12.5 for IgA, and from 1/10 to 1/2.5 for IgE. The wells were subsequently incubated with mouse anti-sheep IgM, IgG, IgA, or IgE diluted 1/400 and, alkaline phosphatase–conjugated goat anti-mouse diluted 1/3000 and developed with PNPP. All incubations were at 37°C, with 100 µl per well, and all dilutions were made in PBS containing 1% Elk and 0.3% Tween-20.

To determine the effect of periodate, which oxidizes and inactivates most carbohydrate antigens, on the antigenicity, ES was coated and treated as described and incubated with 10 µg/ml biotinylated concanavalin A, wheat germ agglutinin, lentil agglutinin (Pierce) or a monoclonal antibody specific for the LDNF antigen (SmLDNF1; Nyame et al., 2000Go). The wells were subsequently incubated with streptavidin–horseradish peroxidase (Pierce) or streptavidin–horseradish peroxidase–conjugated rabbit anti-mouse (Dako) and developed with 0.01% 3,3'5,5'-tetramethyl benzidine dihydrochloride as described elsewhere (Vervelde et al., 2001Go). After 15 min incubation at room temperature, the reaction was stopped with 100 µl 1 M H2SO4. The absorbance was measured at 450 nm.

To determine LDNF-specific antibodies, plates were coated with 2 µg/ml fucosylated LacdiNAc coupled to bovine serum albumin (BSA; Nyame et al., 2000Go), blocked with 5% Elk in PBS, and incubated with sera diluted 1/10. The wells were subsequently incubated with mouse anti-sheep IgM or IgG, alkaline phosphatase–conjugate and developed with PNPP as described. All steps were done with 50 µl per well and incubated at room temperature for 1 h; after each step plates were washed six times. To correct for nonspecific binding, the BSA control surface was used as blank. As positive control we used a monoclonal antibody anti-LDNF (SmLDNF1).

Immunoblots
Extracts of H. contortus were prepared as described previously (Schallig et al., 1994Go). Protein extracts (5–15 µg per lane) were separated by sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) under reducing conditions on a 15% polyacrylamide gel, using the Mini-Protean II system (BioRad, Hercules, CA) and blotted onto a nitrocellulose membrane. The membrane was blocked in a solution of 5% BSA in PBS for 2 h, followed by incubation with monoclonal antibody anti-LDNF (SmLDNF1) in PBS containing 1% BSA for 1 h. After washing in PBS containing 0.1% Tween and incubation with alkaline phosphatase–conjugated goat anti-mouse IgG/IgM, bound antibodies were detected using x-phosphate/5-bromo-4-chloro-3inodyl-phosphate (Boehringer Mannheim, Mannheim, Germany) and 4- nitroblue-tetrazoliumchloride (Boehringer Mannheim).

Mucus antibody levels
Abomasal folds were collected at necropsy and stored at -20°C. The folds were partially defrosted. Mucus was scraped of using a glass objective slide, collected in 3 ml of cold PBS containing a cocktail of protease inhibitors (Complete Mini, Roche, Mannheim, Germany), and kept on ice. Samples were shaken vigorously for 1 h at 4°C, and spun for 30 min 2000 x g at 4°C. The supernatant was transferred to an Eppendorf tube and spun for 30 min 20,000 x g at 4°C. The supernatant was collected and stored at -20°C. Protein concentration was determined using the Bradford assay, and samples were adjusted to 0.25 mg/ml and used undiluted in the ES-specific ELISA as described. To determine ES-specific mucus IgE, the samples were treated for 1 h at 56°C.

To ensure that we did not include lamina propria tissue, the folds were fixed overnight in modified Bouin after scraping, transferred to 70% ethanol, and processed to wax. Sections (3 µm) were cut, rehydrated, and stained with hematoxylin.

Statistical analysis
Statistical analysis was carried out using SPSS statistical package 9.0. Differences between the groups of lambs were tested with the Mann-Whitney test. Correlations between parasitological parameters and antibody levels were tested with Pearson's correlation coefficient. Comparisons were made between the lambs only (groups 2–5) to be able to discover an effect of the adjuvant without having an age-related effect.


    Acknowledgements
 
We thank Kirezi Kanobana, Margreet van der Veer, and Wim Hendrikx for technical assistance. This research was supported by a grant of the Dutch Technology Foundation (STW, Netherlands; project UDG 55.3762, L.V.) and a grant (AI47214) from the National Institutes of Health (R.D.C.).


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: l.vervelde{at}vet.uu.nl Back


    Abbreviations
 
BSA, bovine serum albumin; DDA, dimethyl dioctadecyl ammonium bromide; ELISA, enzyme-linked immunosorbent assay; ES, excretory/secretory glycoproteins; LDN, LacdiNAc; LDNF, GalNAcß1,4(Fuc{alpha}1,3)GlcNAc; PBS, phosphate buffered saline; PNPP, p-nitrophenyl phosphate disodium salt; SDS–PAGE, sodium dodecyl sulfate– polyacrylamide gel electrophoresis


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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