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IMMUNOLOGY, HEALTH AND DISEASE |
-Gal-Conjugated Antigen in Animals with Preexisting High Levels of Natural Antibodies Binding -Gal ResiduesImmunology Section, Adaptation Physiology Group, Department of Animal Sciences, Wageningen University, Marijkeweg 40, 6709 PG Wageningen, the Netherlands
1 Corresponding author: Henk.Parmentier{at}wur.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Gal residue (Gal1-3Galβ1-4GlcNAc) are found in poultry (and humans), which is probably reflected by high levels of natural agglutinating antibodies (Ab) to rabbit red blood cells (RRBC) in plasma from chickens (and humans). Recently, it was shown that -Gal conjugation of proteins induced higher antiprotein Ab responses in -Gal knockout mice, suggesting immune-enhancing features of preexisting Ab binding carbohydrate-protein conjugates. We challenged chickens s.c. with either -Gal-conjugated human serum albumin (HuSA), β-Gal-conjugated HuSA, or unconjugated ("native") HuSA, respectively, and measured primary and secondary Ab responses to HuSA, including isotype IgM and IgG responses, and cellular immune responses in vitro (lymphoproliferation) to HuSA or concanavalin A. -Gal conjugation, but not β-Gal conjugation, of HuSA resulted in significantly decreased primary and secondary Ab responses to HuSA, especially IgG isotype responses, as compared with Ab responses to native HuSA. Lymphoproliferation in vitro was also decreased, although not significantly, in birds challenged with -Gal-conjugated HuSA. High levels of agglutinating Ab levels to RRBC and NAb binding porcine thyroglobulin were detected in all birds, as was true for (natural) Ab levels binding -Gal-conjugated HuSA before immunization, whereas low levels of preexisting (natural) antibodies directed to native HuSA were present in plasma before immunization. Levels of RRBC agglutinins and Ab binding thyroglobulin were not affected by immunization with HuSA, -Gal-conjugated HuSA, or β-Gal-conjugated HuSA. Our data confirm the presence of high levels of (preexisting) NAb in the plasma of chickens directed to the -Gal residue. The decreased responsiveness to -Gal-bearing antigens in the current study shows that, in addition to immune-enhancing features, NAb may also have suppressive effects on specific immune responses, which substantiates the regulatory role of innate immunity (NAb) in mounting specific immune responses.
Key Words: -Gal • human serum albumin • chicken • immune modulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Natural Ab are present in chickens as well (Jalkanen et al., 1983). Chicken NAb bind ovarian antigens (Barua and Yoshimura, 2001), major histocompatibility complex (MHC) class IV (B-G) antigens (Longenecker and Mosmann, 1980; Neu et al., 1984), and various nonself exogenous model glycoproteins and self antigens (Jalkanen et al., 1983; Parmentier et al., 2004), with increasing levels during aging of the birds. Passive adoptive transfer of NAb enhanced specific Ab responses in recipients, suggesting that NAb indeed are involved in enhancing subsequent specific immune reactivity (Lammers et al., 2004). High levels of (agglutinating) NAb binding rabbit red blood cells (RRBC) are present in chicken plasma, which may reflect binding to the Gal1-3Galβ1-4GlcNAc (-Gal) residue (Cotter et al., 2005). In addition, NAb directed to thyroglobulin were detected in chicken plasma (Parmentier et al., 2004), which may be related to the presence of -Gal residues in nonprimate thyroglobulin (Kirkeby and Moe, 2002). In addition, a genetic relation between RRBC agglutinins and NAb binding thyroglobulin on the one hand, and specific Ab responses directed to sheep red blood cells (SRBC) on the other hand was reported (i.e., higher levels of RRBC agglutinins; Cotter et al., 2005), and NAb binding thyroglobulin (Parmentier et al., 2004) were found in chickens divergently selected for higher specific Ab responses to SRBC (high line) as compared with control nonselected birds or birds selected for low Ab responses to SRBC (low line) during subsequent generations, with a correlation between specific SRBC and natural RRBC titers of 0.43 in the 19th generation of divergent selection (Cotter et al., 2005). The presence of agglutinins to RRBC in the low line suggested, however, an important function of NAb binding the -Gal epitope in the viability of chickens.
Recently, it was shown that -Gal conjugation of the HIV gp-120 protein induced high immune responses in -Gal knockout mice to the gp-120 protein, suggesting an immune-enhancing effect of this carbohydrate residue (Abdel-Motal et al., 2006) in combination with induced preexisting Ab directed to -Gal residues. Immunization of (1,3)galactosyltransferase knockout mice (GT0 mice), which, like humans, produce -Gal-specific Ab binding BSA conjugated to -Gal (-Gal-BSA), also led to significant production of anti-BSA IgG Ab without the need for adjuvant, enhancement of BSA-specific cellular immunity, and antiviral T-cell responses after vaccination (Benatuil et al., 2005). These results suggest that preexisting anti--Gal Ab increase the immunogenicity of antigens expressing the -Gal epitope (Benatuil et al., 2005), providing an adjuvant effect that allows more efficient T-and B-cell priming to -Gal-expressing antigens.
In the present study, the effect of -Gal conjugation of protein on the immune response of poultry towards the protein was studied. Further evidence is presented for the presence of high levels of preexisting natural Ab binding the -Gal residue in chickens, which may explain 1) the high levels of RRBC agglutinins and NAb binding thyroglobulin, and 2) the decreased immune responses to -Gal-conjugated proteins as determined in the present study. Our data suggest an important role of NAb binding -Gal (but not β-Gal) in innate regulation of specific immunity. These findings may have important consequences for vaccine management and disease resistance.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents
Gal1-3Galβ1-4(3-deoxyGlcNAc)-Human serum albumin (-Gal-HuSA, 3-Atom Spacer) and Galβ1-3GalNAc-HuSA (β-Gal-HuSA, 3-Atom Spacer) were purchased from Dextra Laboratories (Reading, UK). An average of 8.2 (-Gal) or 7.8 (β-Gal) carbohydrate molecules were present on each mole of HuSA. According to the manufacturer, the protein was not denatured by the carbohydrate conjugation. "Native" HuSA (lot 8763) and (porcine) thyroglobulin were from Sigma-Aldrich Inc. (St. Louis, MO).
Experimental Design
At 12 wk of age (i.e., d 0 of the experiment) and 5 wk thereafter, hens received s.c. either 0.1 mg of HuSA (10 birds), 0.1 mg of -Gal-HuSA (10 birds), or 0.1 mg of β-Gal-HuSA (10 birds), respectively, dissolved in 1 mL of PBS, pH 7.2. Before the first immunization with (carbohydrate-conjugated) HuSA and at 3, 7, 10, 14, 21, and 28 d after primary immunization, blood was taken from the chickens. Similarly, blood was taken at d 35 after primary immunization and before secondary immunization, and at d 3, 7, 10, 14, and 21 after secondary immunization with HuSA (i.e., at 38, 42, 45, 49, and 56 d after primary immunization). Plasma was stored at –20°C until use. Body weight of the chickens was recorded at 17 and 21 wk of age, from which growth during this period was calculated.
Humoral Immune Response to HuSA, -Gal-Conjugated HuSA, and Thyroglobulin
Total Ab titers (IgT) to HuSA, -Gal-conjugated HuSA, and thyroglobulin, respectively, in the plasma from all birds were determined by ELISA at d 0, 3, 7, 10, 21, and 28 after primary immunization, and at d 0, 3, 7, 10, 14, and 21 after secondary immunization with (carbohydrate-conjugated) HuSA. Briefly, 96-well plates were coated with 4 µg/mL of native HuSA or -Gal-HuSA or with 5 µg/mL of thyroglobulin. After subsequent washing with H2O containing 0.05% Tween, the plates were incubated with serial dilutions of plasma. Binding of total chicken Ab to HuSA was detected by using 1:20,000 diluted rabbit antichicken IgGH+L coupled to peroxidase (RACh/ IgGH+L/PO, Nordic, Tilburg, the Netherlands). Plasma samples obtained before primary immunization with (carbohydrate-conjugated) HuSA (d 0 samples) were tested for the presence of (natural) Ab binding HuSA, -Gal-HuSA, or β-Gal-HuSA. Therefore, 4 µg/mL of -Gal-HuSA or β-Gal-HuSA was coated onto the plates, and procedures were as described above. In addition, IgM and IgG Ab binding to HuSA, respectively, were determined at all moments. After incubation with serial dilutions of plasma, plates were incubated with 1:20,000 diluted goat antichicken IgM (GACh/IgM, batch A30-104P-15, Bethyl, Montgomery, TX) coupled to peroxidase, or goat antichicken IgGFc (GACh/IgGFc) coupled to peroxidase (batch A30-102P-20, Bethyl). After washing, tetra-methylbenzidine and 0.05% H2O2 were added and incubated for 10 min at room temperature. The reaction was stopped with 1.25 M H2SO4. Extinctions were measured with a Multiscan instrument (Labsystems, Helsinki, Finland) at a wavelength of 450 nm. Titers were expressed as the log2 values of the dilutions that gave an extinction closest to 50% of Emax, where Emax represents the highest mean extinction of standard positive (pooled) plasma present on every microtiter plate.
Humoral Immune Response to RRBC
Levels of agglutinating NAb binding RRBC were determined as described earlier (Cotter et al., 2005) with minor modifications. Briefly, 50 µL of 1% packed RRBC was mixed 1:2 stepwise with PBS pH 7.2-diluted plasma. Plates were incubated at room temperature for 4 h, after which they were examined for agglutination. Wells not containing plasma served as negative controls; the column number of the last well showing clear-cut evidence of agglutination was recorded as the titer. No qualitative differences in RRBC agglutination patterns were taken into account.
In Vitro Lymphoproliferation to Carbohydrate-Conjugated HuSA and Concanavalin A
In vitro cellular immunity to native HuSA, -Gal-HuSA, β-Gal-HuSA, and the mitogen concanavalin A (Con A) was determined by a lymphocyte stimulation test. On d 28 after primary immunization with carbohydrate-conjugated HuSA, aliquots of 200 µL per well of whole blood diluted 1:60 in RPMI tissue culture medium supplemented with 2.5 x 10–5 M β-mercaptoethanol, 10 µg/mL of streptomycin and 100 IU/mL of penicillin, and 2 mM L-glutamine, in 96-well flat-bottomed plates, and either 5 µg/mL of Con A, 10 or 50 µg/mL of HuSA, 10 or 50 µg/mL of -Gal-conjugated HuSA, or 10 or 50 µg/ mL of β-Gal-conjugated HuSA, respectively, were cultured for 72 h at 41°C and 5% CO2 in a humidified atmosphere. In the last 12 h before harvesting, cultures, set up in triplicate, were pulsed with 0.5 µCi of methyl-3H-thymidine (ICN, Aurora, OH). 3H-Thymidine uptake was determined with a β-scintillation counter (Beckman Coulter, Mijdrecht, the Netherlands). The stimulation index (SI) was calculated as counts per minute (cpm) in mitogen- or antigen-stimulated wells/cpm in unstimulated wells.
Statistical Analysis
Primary and secondary total Ab responses to HuSA, -Gal-conjugated HuSA, and thyroglobulin, primary and secondary isotype Ab (IgM and IgG) responses to HuSA, and titers of agglutinins to RRBC were analyzed by 2-way ANOVA for the effect of treatment (carbohydrate conjugation of HuSA), time, and their interactions by using the repeated measures procedure, with a bird nested within (HuSA type) treatment option. Body weight gain (growth) per sample moment was analyzed by 1-way ANOVA for the effect of HuSA type treatment, as was true for lymphoproliferation (SI) on d 28 after primary immunization with (carbohydrate-conjugated) HuSA. All analyses were according to SAS Institute (1990) procedures. Mean differences between (HuSA type) treatments were tested with Bonferroni’s test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Gal-HuSA, and β-Gal-HuSA-Gal-HuSA [average 8.54 ± 0.38 (SE)] were found in all birds, as was true for titers of total Ab binding β-Gal-HuSA (5.22 + 0.95) in d-0 plasma samples obtained before immunization with carbohydrate-conjugated HuSA or native HuSA, respectively. Much lower titers of total Ab binding native HuSA were detected before immunization with HuSA (2.12 ± 0.38). Natural Ab titers directed to -Gal-HuSA were of both the IgM and IgG isotypes.
Kinetics of Ab Responses to HuSA After Sensitization with HuSA, -Gal-Conjugated HuSA, or β-Gal-Conjugated HuSA
Figure 1
shows the kinetics of the total Ab titers (Figure 1A) and the isotypes IgM (Figure 1B) and IgG (Figure 1C) to HuSA of birds s.c. challenged with HuSA, -Gal-conjugated HuSA, or β-Gal-conjugated HuSA, respectively. The highest titers of all isotypes were found at 7 d after primary immunization and at 7 d after secondary immunization with HuSA, -Gal-conjugated HuSA, or β-Gal-conjugated HuSA. Secondary titers of total Ab and IgG binding HuSA, but not IgM, were higher, although not significantly, than primary titers for all treatment groups. The differences between secondary total and iso-type titers and primary total and isotype titers to HuSA were of similar magnitudes in all treatment groups.
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-Gal-Conjugated HuSA, or β-Gal-Conjugated HuSA-Gal-HuSA, or β-Gal-HuSA were not significantly affected by treatment (Table 1; P > 0.05). There were also no significant contrasts among the 3 treatment groups for IgT and IgM Ab titers at separate sample moments, but a significant contrast between sensitization with HuSA or with -Gal-HuSA was found for IgG directed to HuSA at d 7 postsensitization (Figure 1C
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-Gal-Conjugated HuSA, or β-Gal-Conjugated HuSA; P < 0.05). The highest average titers of total Ab were found in the HuSA-immunized birds, which differed significantly from the -Gal-HuSA-immunized birds, whereas the β-Gal-HuSA-immunized birds were in between. Secondary IgM and IgG responses to HuSA were also affected by a time x treatment interaction, but only for IgG was a contrast found for HuSA immunization vs. immunization with -Gal-HuSA, whereas immunization with β-Gal-HuSA was in between. Significantly higher titers to HuSA were found on d 7 (IgT, IgM, and IgG) and on d 7, 10, and 14 (IgT and IgG) after secondary immunization with HuSA as compared with immunization with -Gal-HuSA (Figure 1).
Kinetics of Ab Responses to -Gal-Conjugated HuSA After Sensitization with HuSA, -Gal-Conjugated HuSA, or β-Gal-Conjugated HuSA
Figure 2 shows the kinetics of the total Ab titers to -Gal-conjugated HuSA of birds s.c. challenged with HuSA, -Gal-conjugated HuSA, or β-Gal-conjugated HuSA, respectively. The highest titers of all isotypes were found at 7 d after primary immunization and at 7 d after secondary immunization with HuSA, -Gal-conjugated HuSA, or β-Gal-conjugated HuSA.
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-Gal-Conjugated HuSA After Sensitization with HuSA, -Gal-Conjugated HuSA, or β-Gal-Conjugated HuSA-Gal-conjugated HuSA during the whole observation period (4 wk) after primary immunization with HuSA, -Gal-HuSA, or β-Gal-HuSA were not significantly affected by treatment (Table 1; P > 0.05). There were also no significant contrasts among the 3 treatment groups for total Ab titers at separate sample moments.
Secondary Ab Responses to -Gal-Conjugated HuSA After Sensitization with HuSA, -Gal-Conjugated HuSA, or β-Gal-Conjugated HuSA
Average total Ab titers to -Gal-conjugated HuSA during the whole observation period (3 wk after secondary immunization) were not significantly affected by treatment (Table 1; P < 0.05). There were also no significant contrasts among the 3 treatment groups for total Ab titers at separate sample moments.
Ab Responses to Thyroglobulin
Mean total Ab responses to thyroglobulin after primary and secondary immunization with either HuSA, -Gal-HuSA, or β-Gal-HuSA, respectively, are shown in Table 2. A significant time x treatment effect was found only after secondary immunization (Table 2).
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-Gal-HuSA, or β-Gal-HuSA, respectively, are shown in Table 2. No significant treatment effect or time x treatment interactions were found.
In Vitro Cellular Immunity
Peripheral blood leukocytes present in whole blood from all birds on d 28 post primary sensitization proliferated in the presence of 5 µg/mL of Con A. Numerically lower responses to Con A were found in birds immunized with -Gal-HuSA (SI 2.2, on average) as compared with birds immunized with HuSA (SI 23, on average), whereas birds immunized with β-Gal-HuSA were in between (SI 10, on average). However, because of the large variation among individual birds (SE 9.8), these differences were not significant. In addition, no significant effects of immunization on T-cell responses to HuSA or carbohydrate-conjugated-HuSA were found (data not shown).
BW (Gain)
Body weights at 17 and 21 wk of age did not differ significantly among the 3 groups: 1,525, 1,392, and 1,534 g at 17 wk for chickens immunized with HuSA, -Gal-HuSA, or β-Gal-HuSA, respectively, and 1,515, 1,450 and 1,618 g at 21 wk for these groups. A contrast (P < 0.05) was found for growth between birds immunized with HuSA (–10 g) and birds immunized with -Gal-HuSA (83 g), whereas growth of the β-Gal-HuSA-immunized birds (58 g) was in between.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Gal-HuSA, or β-Gal-HuSA, respectively. A 10 times lower dose than usual (0.1 vs. 1 mg) of these antigens was used to measure the expected enhancing features of carbohydrate conjugation of the HuSA protein, whereas adjuvants were not applied. Human serum albumin was chosen as the antigen, because, to our knowledge, native HuSA does not contain -Gal residues. Finally, β-Gal-conjugated HuSA was studied as a control for molecular configuration of the -Gal-conjugated HuSA. Specific anti-HuSA Ab titers were measured, because we expected that we could not distinguish anti-HuSA and anti-carbohydrate-conjugated HuSA Ab responses after immunization. High levels of (natural) Ab directed to -Gal-HuSA and, to a lesser degree, also β-Gal-HuSA were detected in the plasma of all birds at 12 wk of age on d 0 of the experiment before immunization, which, with respect to -Gal, corresponded to the high levels of RRBC agglutinins and (natural) Ab titers directed to porcine thyroglobulin before and after immunization. Because RRBC agglutinins and Ab titers to thyroglobulin were not affected by the immunizations, we expected that a change in Ab titers directed to -Gal residues was unlikely. Indeed, Ab responses to -Gal-conjugated HuSA did not differ among the 3 treatment groups, whereas the increase in these titers to -Gal-HuSA after primary and secondary immunization with (conjugated) HuSA was also much lower compared with the Ab responses to native HuSA, and was probably also not initiated by the -Gal residues, as can be concluded from the lack of responses to RRBC and thyroglobulin. Body weights and growth did not reveal negative consequences of immunization with carbohydrate-conjugated HuSA.
Natural antibodies may act as the specific humoral component of innate immunity in cooperation with the immune system. Various specific immune responses, both humoral (Tomer and Shoenfeld, 1988; Thornton et al., 1994; Ochsenbein et al., 1999; Lammers et al., 2004) and cellular (Stäger et al., 2003), of nature are enhanced by, or are positively correlated with, high levels of NAb in mammals and chickens. In addition, higher levels of NAb are found in birds selected for high specific Ab responses to SRBC (Parmentier et al., 2004; Cotter et al., 2005), which is not based on antigen cross-reactivity (H. K. Parmentier, unpublished results). Natural Ab binding -Gal residues may act as a reservoir for immunity, and have also been proposed to represent an essential requirement for immune reactivity, based on the observation that birds selected for low specific Ab responses to SRBC also show NAb directed to various antigens (Parmentier et al., 2004; Cotter et al., 2005). Measuring NAb levels in birds (e.g., via estimating RRBC agglutinins) may provide estimates not only of innate immunity, but also of specific immunity and fitness (Matson et al., 2005). Natural Ab levels were indicative of the survival of chickens during a laying period (Star et al., 2007). In addition to chickens (McKenzie et al., 1999), humans and other primates also lack the -Gal-residue on erythrocytes, which explains the high levels of anti--Gal Ab in humans (1 to 8% of IgG). Anti--Gal Ab production is probably initiated by -Gal epitopes present on the intestinal microbiota. These anti--Gal Ab may play an important role in protection toward -Gal-residue-expressing pathogens (McKenzie et al., 1999; Abdel-Motal et al., 2007; Kim et al., 2007) and other pathogens (Briles et al., 1981; Casali and Schettino, 1996; Haury et al., 1997; Reid et al., 1997; Martin et al., 2001). Anti--Gal Ab, on the one hand, account for the rapid expulsion of xenotransplants (Galili, 1993), but on the other hand, they facilitate immune responses to -Gal-bearing tumor vaccines in humans (Galili and LaTemple, 1997), probably by targeting dendritic antigen-presenting cells. Most mammals (but not humans and primates) do contain -Gal residues on their erythrocytes, and therefore do not contain these high levels of anti--Gal Ab as in humans and chickens. Knockout mice, either for -Gal expression or enzymes involved in -Gal expression, mount high Ab responses to proteins conjugated with -Gal (Benatuil et al., 2005; Abdel-Motal et al., 2006), suggesting high immunogenic properties of -Gal residues and an adjuvating effect of preexisting anti--Gal Ab. Finally, anti--Gal Ab responses are probably T-cell dependent (Cretin et al., 2002). Taking this into account, we expected enhanced specific humoral and cellular immune responses to HuSA in chickens immunized with -Gal-conjugated HuSA. Surprisingly, the present study with chickens, which, unlike most mammals, do have natural preexisting high levels of NAb binding -Gal-residues, revealed that -Gal conjugation, and to some minor extent also β-Gal conjugation, decreased specific immune responses to HuSA. In this respect, it should be noted that chickens were not presensitized with -Gal residues, as opposed to the knockout mouse models. In addition, levels of agglutinins to RRBC were not affected. In particular, both primary and secondary (T-cell dependent) IgG responses to HuSA were 8-fold lower in -Gal-HuSA-immunized birds (Figure 1C), which cannot be explained by immunization doses being too high. It is not unreasonable to suggest that the high levels of preexisting Ab binding -Gal-HuSA may account for the decreased Ab responses to HuSA; that is, the high levels of NAb to -Gal in this respect were not enhancing, but may either have accelerated clearance of -Gal-HuSA or prevented activation of HuSA-specific B cells because of the masking of HuSA. The similar magnitudes of differences between secondary and primary titers in all treatment groups suggest that the capacity to mount T-cell-dependent Ab responses to HuSA was not affected by carbohydrate conjugation of HuSA. Secondary immune responses to haptens can be either suppressed or augmented, depending on the titer and isotype of preexisting Ab. Augmentation may be based on binding to new antigenic epitopes formed by coupling of the hapten to the carbohydrate residue, whereas masking of antigens may account for suppression of secondary responses (Haughton and Makela, 1973; Benatuil et al., 2005). In mammals, the outcome of Ab feedback regulation enhancement or suppression also depends on the nature of the -Gal-conjugated antigen (particulate vs. soluble), the isotype of the preexisting Ab, the type of Fc and complement receptors involved, and the moment that Ab act during an immune response. The fact that NAb are usually IgM may help to initiate Ab responses (reviewed by Getahun and Heyman, 2006). Further studies are required to elucidate these mechanisms in poultry. In this respect, little is known of the effect of carbohydrate-protein coupling ratios and dose effects. However, when high levels of preexisting anti--Gal residue NAb prevent immune activation, this may have important consequences for health management. On the one hand, these NAb may act as an initial first protection to -Gal-residue-bearing pathogens; on the other hand, such NAb may negatively interfere with immune protection of vaccines containing -Gal residues. Future studies should address these issues as well as the role of -Gal in resistance to infection in poultry. Finally, one must keep in mind that -Gal conjugation of soluble proteins may not reflect the immune-activating features of -Gal-expressing particulate microbes.
The origin and induction of NAb are the subject of debate. The repertoire and levels of NAb may either be shaped by continuous polyclonal stimulation by exogenous microbes initiating cross-reactivity-driven responses of autoreactive B cells, or correspond with the secretion of naturally occurring autoreactive B-cell clones, or both (Avrameas et al., 1983). The higher levels of NAb in plasma from old chickens corresponds to the idea that exogenous stimuli enhance the formation of NAb (Prokesova et al., 1996), which may account for NAb binding -Gal residues. On the other hand, the (enhanced) binding of plasma Ab to ovalbumin and the binding of tissue antigens correspond to the proposed naturally occurring B-cell reactivity. Axenic, nude, or newborn mice do not exhibit natural Ab levels different from mice bred under standard conditions, and hybridomas from nonimmunized mice include autoreactive B-cell clones in the absence of autoimmune pathology (Quan et al., 1997; Boes, 2000). The presence of high levels of polyreactive NAb in the serum and mucosa from mammals and birds and the high frequency of "autoimmune" idiotypes suggest important effector and regulatory functions of NAb (Quan et al., 1997). Most NAb are polyreactive to phylogenetically conserved structures: nucleic acids, heat shock proteins, carbohydrates, phospholipids (Boes, 2000), and (autologous) ABO blood group antigens (Spalter et al., 1999). Apart from binding to evolutionary conserved molecules inside cells (actin, myosin, tubulin, and DNA), NAb also bind CD5, CD4, CD8, Ab, MHC (Berczi et al., 1998), 
-interferon (Caruso and Turano, 1997), and interleukin-2 (Balsari and Caruso, 1997), arguing for an immunoregulatory role for NAb. These NAb do not interfere with the biological activities of "exogenous antigens," and may thus function within the immunoregulatory processes by limiting the intensity, duration, or both of immune responses.
From the present data, we conclude that high levels of (natural) Ab binding -Gal residues, with which the animal has not been immunized before, are present in poultry. Although many questions remain, the present study suggests that high levels of preexisting (natural) Ab may perform an important role in immune responsiveness other than enhancing specific immunity. Conjugation of antigen with -Gal did not enhance but decreased specific immunity in animals with high preexisting levels of NAb binding -Gal. Acute clearance of the antigen because of high NAb levels may have prevented the subsequent activation of specific immunity, which may not only reflect a form of functional innate immune resistance, but also a form of innate immune regulation of specific immunity. Experiments are in progress to address these issues.
Received for publication November 30, 2007. Accepted for publication January 20, 2008.
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), 
), or β-Gal-HuSA (
) at 12 wk (primary immunization) and 17 wk of age (secondary immunization). a-cMeans with different letters per sample time point differ (P < 0.05). The arrow indicates the moment of secondary immunization.