CHAPTER 8 IMMUNOLOGICAL IMPLICATIONS OF PEPTIDE CARBOHYDRATE MIMICRY

CHAPTER 8 IMMUNOLOGICAL IMPLICATIONS OF PEPTIDE CARBOHYDRATE MIMICRY

Immunological Implications of Peptide-Carbohydrate Mimicry 8.1 Introduction The two chemically dissimilar molecules, a peptide (12mer) and a carbohydrate moiety (methyl a-d-mannopyranoside), sharing a common receptor, Concanavalin A, were earlier shown to exhibit molecular mimicry. The polyclonal antibodies raised against sugar show crossreactivity with peptide and its various analogs and the antibodies against the peptide crossreact with sugar (Kaur et al., 1997). The mimicry between peptide and carbohydrate ligands was further probed at the structural level, which is extensively described in earlier chapters. Here, we have further attempted to asses the functional mimicry between peptide and sugar as a function of progression of the immune response so as to understand the course of maturation of the mimicry and also to comprehend various factors affecting it. 8.2 Results 8.2.1 Tile Mimicry Analyzed at Different Levels of Antibody Response Earlier experiments have indicated that immunization of Balb/c mice with peptide or sugar conjugate elicits crossreacting antibodies (Kaur et a/., 1997). It was important to examine the issues related to the extent of crossreadivity at different time points during the course of maturation of the antibody response. We have analyzed the induction and subsequent progression of the mimicry at different levels of antibody maturation. 8.2.1.1 JgM Response Two separate groups of female Balb/c mice were immunized with Single dose (1fl9 protein/mouse) of sugar and peptide conjugated to BSA (bovine serum 14

.5 ----l_'-- ~ Sugar binding Peptide binding.4 E c CJ) -.::t Ctl.3.2.1.------ Control Anti Anti sugarab peptideab Figure 8.1: Analysis of mimicry at IgM level. The polyclonal antisugar antibody response at IgM level is dominated by crossreacting antibodies. The control for crossreactivity is shown.

Immunological Implications of Peptide-Carbohydrate Mimicry albumin), respectively. The serum was collected at regular intervals, starting as early as day5. The serum was tested for IgM specificity against the DT (diphtheria toxoid) conjugated immunogen. The early primary response generated against the antigen could be detected within a week after immunization and reaches a peak at day14. Thus all the subsequent analysis for IgM was done at day14 (Figure 8.1). Both sugar as well as the peptide induced IgM response, although immunogenicity varied widely in the two cases. The initial analysis revealed that anti sugar polyclonal antibody titers are very high. Moreover, almost the entire population of the antisugar antibodies generated, were capable of crossreacting with the peptide. In contrast, the antipeptide polyclonal antibodies show very low titers at IgM level. When these low titer antibodies were analyzed for crossreactivity, they showed negligible binding to sugar. 8.2.1.2 IgG Response The serum collected at regular intervals was also analyzed for IgG specificity. The primary IgG response reaches a peak at day28 and hence all subsequent analysis of primary IgG was done at day28. The mice were further boosted with the corresponding antigen at day42 to analyze the crossreactivity at secondary IgG level. The secondary IgG analysis has been done in all the cases at day56. As evident from Figure 8.2A, the antisugar antibodies show poor primary IgG titers. The booster at day42 enhances the antibody titer upto 1 Of old. The corresponding cross reactivity of antisugar antibodies follows a similar trend both at primary as well as the secondary IgG response. Although, the antisugar antibodies do not show any detectable crossreactivity at day28, the Signal increases Significantly after the booster (Figure 8.28). The antipeptide polyclonal antibodies on the other hand show very high titers against peptide at primary IgG level, such that it 15

A 2. ~ Anti Sugar pabs ~ Anti Peptide pabs E c m -.;t... as 1.5 1..5. Preimmune Primary Secondary B.4 ~ Anti Sugar pabs m Anti Peptide pabs E c m -.;t... m.3.2.1 o.olbj.-- Preimmune Primary Secondary Figure B.2: Maturation of the humoral response. [AJ IgG titers of antisugar and antipeptide pabs as a function of time. [BJ The crossreactivity of antisugar and antipeptide pabs as a function of time.

Immunological Implications of Peptide-Carbohydrate Mimicry reaches saturation and there is no significant increase in the antibody population after the booster (Figure 8.2A). The crossreactivity in case of antipeptide antibodies is higher than in case of antisugar antibodies at day28 and increases significantly after the booster. However, day56 antibodies against peptide and sugar show comparable crossreactivity (Figure 8.28). 8.2.1.3 Competitive Studies The affinities of antisugar and antipeptide antibodies at different levels of maturation were assayed using competitive ELISA. Various peptide and sugar analogs compete with 12mer- and sugar-ot coated on the plate for binding to anti sugar and anti peptide antibodies respectively in solution. The crossreactivity of antisugar antibodies, assayed using direct binding ELISA, shows low signals at the primary response, as the antisugar antibody titers were low. But, the competition amongst various peptides for binding to antisugar antibodies indicates that affinity of these antibodies for mimicking ligands is notable even though the titers are low. The antisugar antibodies at day28 show higher crossreactivity with 1mer and 15mer and in comparison to 12mer. The same antibodies, after booster, at day56, exhibit enhanced crossreactivity with 12mer (Figure 8.3A). Similarly, the antipeptide antibodies also show crossreactivity with different sugar analogs at the primary response. The affinity of the antibodies is highest for methyl a-o-mannopyranoside and lowest for methyl a-oglucopyranoside. However, secondary IgG shows enhanced affinity for glucopyranoside and significantly lower affinity for a-lactose (Figure 8.38). 16

RVWYPYGSYLTASGS A OVFYPYPYASGs~IIII~i:::::::::~~ MYWYPYASGs~IIII~~~~lIlIlIii~;-------~~ ~Primary ~Secondary o 5 1 15 2 25 3 B % Inhibition Me a-o-mannopyranoside m~m~m~mmmm~i~i~ie;;~~--- 35 Me a-o-g/ucopyranoside ~~~~m~ml=== ~Primary ~Secondary o 5 1 15 2 25 3 35 4 % Inhibition Figure 8.3: Competitive binding analysis of antisugar and antipeptide antibodies. fa] The comparison of antisugar antibody crossreactivity with various peptide analogs as a function of time. fb] The comparison of anti peptide antibody crossreactivity with various sugar analogs as a function of time.

Immunological Implications o/peptide-carbohydrate Mimicry 8.2.2 Modifications in T-cell Help 8.2.2.1 Prepriming To explore the role of limiting T-cell help in the process of selection of antibodies showing crossreactivity in case of antisugar antibodies, an experiment was done, where 8alb/c mice were first primed with 8SA before immunization with conjugated sugar. A booster of sugar was given at day42. The rationale here is to ensure the presence of an elevated frequency of 8SA primed Th cells at the time of antigenic challenge. Sera from such mice were collected at day28 and day56 after the first immunization, to assay primary and secondary antibody titers respectively. It was observed that the mice primed with 8SA, show better titers at primary IgG level than those, which were unprimed. As expected, the antibody titers increase after booster. On the other hand, the crossreactivity of the antibodies does not follow a similar trend. The crossreactivity of antisugar antibodies at day28 is higher than in unprimed case and reaches saturation. It shows no increase even after the booster unlike in unprimed case discussed earlier (Figure 8.4). 8.2.2.2 Analysis Using the Single T-cell Epitope In order to compare the extent of crossreactivity of antibodies using promiscuous T-cell epitopes as against the protein, the 8-cell epitope was synthesized colinearly with two known T-cell epitopes to obtain peptides that would be immunogenic in mice. The T-cell epitopes chosen were those that had been previously well characteri7sd and were derived either from residue 44-6 of respiratory syntical virus (designated as RSV) (Nicholas et a/., 1989) or from residues 83-844 of tetanus toxoid (designated as TT) (Ho et a/., 199). In all these cases a spacer of two glycine residues separated the 8- and T-cell epitopes 17

1.4 1.2 ~ Sugar binding ~ Peptide bindirg 1. E c.8 ) ~... m.6.4.2. ~~~~:L-~~~~---I:.<~~=i4...J Prepriming BSA BSA BSA BSA BSA BSA Priming Booster Sugar Sugar Sugar Sugar Sugar Sugar Figure 8.4: Analysis of specificity and crossreactivity of antisugar pabs as a function of time on prepriming.

1.4 1.2 A - coaling Sug-BSA -coating 13mar-BSA.8.7 8 E c > V -co ci cj E c 1..6 > V -co ci.5.8 cj.4.6.3.4 2 4 6 8 1 12 2 4 6 8 1 Days Days c RSV:CEYNVFHNKTFELPRA n : PGINGKAIHLVNNESSE Figure 8.5: Analysis of antipeptide antibody crossreactivity as a function of time using promiscuous T cell epitopes. [A] RSV. [B] IT. [e] Sequence of the two T cell epitopes used.

Immunological Implications of Peptide-Carbohydrate Mimicry sequences. The immunization schedule followed for these antigens was same as for peptide-8sa. The analysis of the primary IgG shows that unlike in case where a carrier protein is used as a T-cell epitope, the overall titers of the antibody are low in this case, however the corresponding crossreactivity is very high (Figure 8.SA and 8). Further, the antibody titers are lower in case of IT as compared to RSV. After the antibody titers fell to basal levels by day42, a booster of the corresponding antigen was given in both the cases. SurpriSingly, after the booster, there was no significant enhancement in the antibody titers in either case showing that the antibodies reach saturation in the primary response itself. The secondary IgG antibodies formed have titers comparable to the primary response. Similarly, the crossreactivity does not show a corresponding increase in the signal. In fact, crossreactivity levets are lower than in the primary response. 8.2.3 Tile Sugar and Peptide are Immunological Mimics The crossreactivity of antibodies with a chemically different molecule as seen above reflects that the two molecules are mimicking at the topological level. In order to see if the immune system is capable of identifying these molecules as mimics, cross-immunization experiments were done. A group of female 8alb/c mice was immunized intra peritoneally with sugar conjugated to 8SA. The serum was collected at regular intervals and analyzed by ELISA for the presence of antigen specific antibodies. The immune response was further boosted by giving the sugar mimicking peptide (12mer) conjugated to 8SA as a booster at day42. The analysis of the primary IgG response shows that the antisugar antibodies reached a peak at day28. It was discovered that primary IgG levels of antisugar antibodies could be amplified on giving chemically different molecule as a booster 18

Priming Booster Day Sugar 28 Sugar Sugar Sugar Peptide 56 Sugar 56..5 1. 1.5 2. 2.5.. at 49 nm Figure 8.6: The IgG response against sugar can be boosted using the carbohydrate mimicking peptide.

Immunological Implications o/peptide-carbohydrate Mimicry (Figure 8.6). The control, group where no booster was given, shows same titers as the day28. This implies that the peptide is capable of reactivating the T-cells that have been earlier primed by the sugar and is capable of recruiting the T-cell help. 8.3 Discussion The analysis of the antisugar and antipeptide antibody specificity and crossreactivity at different time points revealed certain differences. Whereas the IgM response, in case of sugar was dominated by mimicking antibodies, the titers of antipeptide antibodies were low and no mimicry could be detected. Sugar being a small molecule and structurally simpler provides a smaller surface area of contact for the corresponding antibody and hence can be treated as a single epitope. Thus, majority of the antibody population generated against sugar would be capable of showing crossreactivity with mimicking epitope on the peptide. Peptide being a large, and structurally more complex and flexible molecule in comparison to sugar will constitute multiple epitopes and all accessible determinants on the surface of peptide will be recognized. Thus, the early IgM response in this case would be composed of antibody specificity that spanned the entire peptide and only small population of which will crossreact with the sugar. Similarly, differences were observed in the binding profiles at primary and secondary IgG level. The observed differences in IgM and primary IgG response of antisugar and antipeptide antibodies, especially in terms of mimicry could reflect the differential proliferative abilities of the corresponding epitope-specific 8 cells. The results show that the early primary humoral response against sugar is biased in favor of the mimicking epitope. Further the IgM to IgG class switch for antibodies against sugar is less efficient than in case of peptide. Competitive data suggests that in the primary response, sugar elicits low titer but high affinity antibodies capable of showing mimicry with various peptide 19

Immunological Implications of Peptide-Carbohydrate Mimicry analogs. However, no significant change occurs in the affinity of antibodies, eventhough the titers increase significantly after the booster. In case of antipeptide antibodies, the affinity profile against different sugars changes although the titers remain the same. The antisugar antibody response analyzed in terms of titers as well as crossreactivity at various time points by modifying the T-cell help gave interesting results. The antisugar antibody titers at primary immune response level improved on preprimimg as compared to the unprimed case. Thus, in presence of unlimited T-cell help, all the B-cell clones get activated and therefore we see increase in the mimicry signal even at primary IgG level. However, the mimicry reaches saturation at the primary response and shows no increase after the booster. Further more, use of single T-cell epitope, as against a protein carrier, influences the immune response in terms of titers. The overall signal for peptide coupled to promiscuous T-cell epitopes is lower than peptide coupled to a carrier, however the mimicry follows a reverse trend. This may be because the B-cells that are activated using the single T-cell epitope are fewer in comparison to the protein carrier. This is expected because the size of the carrier being large will activate larger numbers of B-cells as against using a single T-cell epitope. However, the numbers of clones of the B-cells that yield mimicking antibodies against the sugar are higher. In other words, the immunodominant epitope gets defined much early in the course of maturation of the immune response and hence we see higher levels of mimicry in both the cases at primary response itself. Thus, when we use a single T- cell epitope, the mimicry signal dominated in the early IgG response as the selection seems to have occurred in the primary response itself and on subsequent maturation the mimicking antibodies did not get selected. The mimicry between chemically different molecules is not just manifested in 11

Immunological Implications o/peptide-carbohydrate Mimicry terms of crossreactivity of polyclonal antibodies and hence is not a physical phenomenon. The antisugar antibodies could be boosted using a carbohydrate mimicking peptide on cross immunization. The two molecules are recognized as mimics even by the immune system. Thus, mimicry is also elicited in terms of reactivating the memory T-cells. To conclude, the molecular mimicry observed between the two molecules manifests in the immune response. The primary IgG response against one molecule can be enhanced using the mimic. However the maturation of the humoral response analyzed in terms of primary (lgm and IgG) and secondary (lgg) antibody titers, shows subtle differences in their and crossreactivity profiles against the two ligands. The antigenic immunodominance during maturation may be invoked to explain these differences. The matured polyclonal antibody response against any ligand need not necessarily represent a complete topological map of the antigen. Therefore, the extent of mimicry may depend on the extent of overlap between the immunodominant epitope and the mimicking epitope. It was observed that the mimicry as seen by the immune system, depends on the extent and nature of the T- cell help provided either in terms of a carrier protein or the single peptide as the T- cell epitope. Thus, the carbohydrate-peptide mimicry, which was originally defined as a static topological equivalence between two chemically distinct molecular species in terms of polyclonal antibody crossreactivity (Kaur et a/., 1997) is influenced by the kinetic immunological factors. This observation may provide important basis for analyzing mechanisms responsible for autoimmune disorders. III