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Infection and Immunity, November 2005, p. 7311-7316, Vol. 73, No. 11
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.11.7311-7316.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Role of Complement Receptor Type 2 and Endogenous Complement in the Humoral Immune Response to Conjugates of Complement C3d and Pneumococcal Serotype 14 Capsular Polysaccharide

Joyce K. Mitsuyoshi, Yong Hu, and Samuel T. Test*

Children's Hospital Oakland Research Institute, Oakland, California 94609

Received 27 May 2005/ Returned for modification 6 July 2005/ Accepted 21 July 2005


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ABSTRACT
 
Conjugation of the complement fragment C3d to both T-cell-dependent (TD) protein and T-cell-independent type 2 (TI-2) polysaccharide antigens enhances the humoral immune response in mice immunized with either type of antigen. However, the ability of C3d-protein conjugates to enhance the antibody response in mice deficient in complement receptor types 1 and 2 (CR1 and CR2) has raised questions about the role of C3d-CR2 interactions in the adjuvant effect of C3d. In this study, we examined the role of CR2 binding and endogenous complement activation in the antibody response to conjugates of C3d and serotype 14 pneumococcal capsular polysaccharide (PPS14). To block binding of PPS14-C3d conjugates to CR2, mice were immunized with a mixture of vaccine and (CR2)2-immunoglobulin G1 (IgG1). Mice receiving (CR2)2-IgG1 at the time of primary immunization had a marked reduction in the primary anti-PPS14 antibody response but an enhanced secondary anti-PPS14 response, suggesting that C3d-CR2 interactions are required for the primary response but can have negative effects on the memory response. Further, compared with mice receiving PPS14-C3d having a high C3d/PPS14 ratio, mice immunized with PPS14-C3d with low C3d/PPS14 ratios had an enhanced secondary antibody response. Treatment of mice with cobra venom factor to deplete complement had insignificant effects on the antibody response to PPS14-C3d. Experiments with CBA/N xid mice confirmed that PPS14-C3d conjugates retain the characteristics of TI-2 rather than TD antigens. Thus, the adjuvant effect of C3d conjugated to PPS14 requires C3d-CR2 interactions, does not require activation of endogenous complement, and is not mediated by TD carrier effects.


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INTRODUCTION
 
In recent years, the ability of complement to enhance the humoral immune response (15, 16) has been put to practical use by employing the complement fragment C3d as an adjuvant in a variety of vaccine constructs (3, 4, 11, 13, 18, 19, 23). Our studies (20) have focused on the use of C3d as an adjuvant in polysaccharide vaccines: specifically, as a component of vaccines against the capsular polysaccharides of Streptococcus pneumoniae, which are T-cell-independent type 2 (TI-2) antigens. We have used the capsular polysaccharide from serotype 14 pneumococcus (PPS14) for these studies, as complement activation has previously been shown to be necessary for an optimal immune response to unmodified PPS14 (12).

The adjuvant effect of C3d in conjugate vaccines has been assumed to involve interactions of the conjugated C3d with CR2 on B lymphocytes and follicular dendritic cells and to bypass the requirement for endogenous complement activation and the consequent covalent deposition of C3d(g) on antigen which follows immunization. The validity of this assumption has been opened to question by the publication of results showing that C3d conjugates can induce an effective antibody response in mice lacking CD21/35 expression (6). One possible explanation that was proposed to explain CR2-independent effects of C3d was that C3d could simply be functioning as a T-cell-dependent (TD) protein carrier (6). The goals of the studies reported here were to determine (i) whether binding to CR2 is a necessary component of the antibody response to PPS14-C3d, (ii) whether conjugation of C3d to PPS14 bypasses the requirement for endogenous complement, and (iii) whether C3d behaves as a TD protein carrier. We also wished to see whether restricting inhibition of CR2 binding or complement activation to the time of primary immunization had different effects on the primary and secondary anti-PPS14 antibody responses to PPS14-C3d, similar to our previous findings for unmodified PPS14 and PPS14-ovalbumin (OVA) (21). Because this is difficult or impossible using C3 or complement receptor knockout mice, we treated mice with a soluble form of CR2 (7) to prevent binding of C3d to cell surface CR2 and used cobra venom factor (CVF) to deplete serum complement prior to immunization.


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MATERIALS AND METHODS
 
Conjugate vaccines. PPS14-C3d and PPS14-OVA were prepared by conjugating purified mouse C3d or OVA monomers to PPS14 as previously described (20, 21). For the experiments portrayed in Fig. 1, 2, and 4, the PPS14-C3d conjugate used had a 6.4:1 molar ratio of C3d to PPS14, assuming a molecular mass of 100,000 Da for PPS14. The PPS14-OVA conjugate had an OVA/PPS14 molar ratio of 4.7:1.



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  		FIG. 1. Effect of treatment with (CR2)2-IgG1 on anti-PPS14 IgM and IgG responses following immunization with PPS14-C3d. BALB/c mice (eight mice per group) were immunized by subcutaneous injection with 0.5 µg of PPS14 as PPS14-C3d with or without the additions listed below. A second injection of PPS14-C3d without any additions was given 42 days after the first (arrows). Serum anti-PPS14 IgM (left) and IgG (right) GMCs are shown for mice immunized with PPS14-C3d alone (Untreated), PPS14-C3d plus 10F7MN murine monoclonal IgG1 anti-human glycophorin A (Control IgG1), PPS14-C3d plus (CR2)2-IgG1 at a 10:1 molar ratio of inhibitor to C3d [(CR2)2-IgG1 low] or PPS14-C3d plus (CR2)2-IgG1 at a 20:1 molar ratio of inhibitor to C3d [(CR2)2-IgG1 high]. Error bars represent the 95% confidence interval. P values for mice immunized with PPS14-C3d plus (CR2)2-IgG1 at the lower ratio versus mice immunized with PPS14-C3d plus control IgG1 were highly significant for all postimmunization bleeds for IgM (P ≤ 0.002) and for all post-primary bleeds for IgG (P ≤ 0.0006). P values for post-secondary IgG anti-PPS14 were 0.17 at 10 days and 0.10 at 25 days. P values for mice immunized with PPS14-C3d plus (CR2)2-IgG1 at the higher ratio versus mice immunized with PPS14-C3d plus control IgG1 were highly significant for all postimmunization bleeds for IgM (P ≤ 0.00004) and IgG (P ≤ 0.003) except for IgG at 10 days post-secondary immunization (P = 0.09).



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  		FIG. 2. Effect of CVF treatment on the anti-PPS14 antibody response in mice immunized with PPS14-C3d. Groups of 10 BALB/c mice were immunized by subcutaneous injection with 0.5 µg of PPS14 as PPS14-C3d. Repeat 0.5-µg immunizations were given at 42 and 84 days after primary immunization (arrows). Serum anti-PPS14 IgM (left) and IgG (right) GMCs are shown for mice receiving no CVF or receiving CVF at the time of primary immunization. Error bars represent the 95% confidence interval. Differences in IgM and IgG anti-PPS14 concentrations between untreated and CVF-treated mice failed to achieve statistical significance at any time before or after immunization.



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  		FIG. 4. Anti-PPS14 antibody response to PPS14-C3d and PPS14-OVA in CBA/N xid mice. Groups of eight CBA/N xid mice were immunized by subcutaneous injection with 1 µg of PPS14 as PPS14-C3d or PPS14-OVA at days 0, 42, and 84. Anti-PPS14 IgM (left) and IgG (right) are shown for individual mice at 25 days after tertiary immunization. Horizontal lines indicate the anti-PPS14 GMC for each group of mice. Anti-PPS14 concentrations were significantly higher for mice immunized with PPS14-OVA compared with mice immunized with PPS14-C3d for both anti-PPS14 IgM (P = 0.0004) and IgG (P = 2 x 10–8).

For the experiment portrayed in Fig. 3, in which PPS14-C3d conjugates having different ratios of C3d to PPS14 were compared, three different PPS14-C3d conjugations were performed and the resulting preparations were subjected to chromatography on Bio-Gel P-300 to remove free C3d (Bio-Rad Laboratories, Hercules, Calif.). PPS14 and C3d concentrations in each conjugate fraction eluting from the column were determined, and three individual fractions having different C3d/PPS14 ratios were selected for use in vaccinations. The PPS14-C3d conjugates used for immunizations had C3d/PPS14 ratios of 2.2:1, 3.0:1, and 6.3:1.



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  		FIG. 3. Anti-PPS14 antibody response after immunization with PPS14-C3d conjugates having high and low C3d/PPS14 ratios. Groups of eight BALB/c mice were immunized by subcutaneous injection with 1 µg of PPS14 as PPS14-C3d using PPS14 conjugates having C3d/PPS14 ratios of 2.2:1 (left), 3.0:1 (middle), or 6.3:1 (right). An identical injection of PPS14-C3d was given 42 days after the first (arrows). Serum anti-PPS14 IgM (top) and IgG (bottom) concentrations are shown, with distinct symbols representing values from different mice. The lines connect the anti-PPS14 GMCs for each bleed. P values for maximal IgM anti-PPS14 GMC at 10 days post-secondary versus 10 days post-primary immunization were 0.03 for the 2.2:1 C3d/PPS14 conjugate ratio, 0.05 for the 3.0:1 C3d/PPS14 conjugate ratio, and 0.77 for the 6.3:1 C3d/PPS14 conjugate ratio. P values for maximal IgG anti-PPS14 GMC at 25 days post-secondary versus 25 days post-primary immunization were 0.01 for the 2.2:1 C3d/PPS14 conjugate ratio, 0.06 for the 3.0:1 C3d/PPS14 conjugate ratio, and 0.68 for the 6.3:1 C3d/PPS14 conjugate ratio.

Soluble CR2. Soluble CR2 in the form of a recombinant (CR2)2-immunoglobulin G1 (IgG1) chimera (7) was purified from culture supernatants of J558L cells expressing the construct (kindly supplied by Ed Hayman, Avant Immunotherapeutics, Needham, Mass., with the permission of Douglas Fearon, University of Cambridge, Cambridge, United Kingdom). Supernatants were collected from cells grown in GIBCO Hybridoma-SFM (Invitrogen Corp, Grand Island, N.Y.), and (CR2)2-IgG1 was purified by chromatography on a HiTrap protein G column (Amersham Biosciences, Uppsala, Sweden). The ability of (CR2)2-IgG1 to inhibit C3d conjugate binding to CR2 was confirmed by flow cytometry using Raji cells as described previously (20). The mouse IgG1 monoclonal antibody 10F7MN against human glycophorin A was similarly purified and used as a control. Final (CR2)2-IgG1 and 10F7MN preparations were suspended in phosphate-buffered saline (PBS) and filtered with a 0.2-µm-pore filter before use.

Mice and immunizations. Female BALB/c mice were from Charles River Laboratories, Hollister, Calif., and used at ages 8 to 11 weeks. Female CBA/J and CBA/N xid mice, ages 9 to 11 weeks, were from The Jackson Laboratory, Bar Harbor, Maine. Groups of 8 to 10 mice were immunized with 0.5 or 1 µg (as polysaccharide) of PPS14-C3d or PPS14-OVA diluted in 200 µl of sterile, endotoxin-free PBS (Sigma Chemical Co., St. Louis, Mo.). Vaccines were administered by subcutaneous injection at days 0 and 42 (and in some cases at day 84), with the total dose divided equally between two sites. Mice were treated with CVF at the time of primary immunization as previously described (21). For experiments in which mice were treated with (CR2)2-IgG1, PPS14-C3d was mixed with either PBS, 10F7MN control mouse IgG1, or (CR2)2-IgG1 at 10:1 or 20:1 molar ratios to C3d and incubated overnight at room temperature prior to immunization. Blood for anti-PPS14 IgM and IgG determinations was obtained 3 days prior to and at 10 and 25 days after each immunization.

ELISAs for determination of anti-PPS14 antibody concentrations. Serum anti-PPS14 antibodies were measured using enzyme-linked immunosorbent assays (ELISAs) specific for anti-PPS14 IgM and anti-PPS14 IgG as described previously (21). Purified 9.2 IgM and 44.2 IgG anti-PPS14 monoclonal antibodies (provided by Alexander H. Lucas, Children's Hospital Oakland Research Institute) were used as standards in the assays.

Statistical analysis. Serum anti-PPS14 IgM and IgG concentrations were determined for individual mice within each immunization group and the geometric mean and 95% confidence intervals of the geometric mean were calculated. To eliminate the effects of mouse-to-mouse variability, statistical comparisons were made on log-transformed data using Student's t test. Statistical significance was set at a P value of ≤0.05.


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RESULTS
 
Effect of soluble CR2 on the anti-PPS14 antibody response to PPS14-C3d. To examine the role of interactions between C3d and CR2 in the anti-PPS14 antibody response to PPS14-C3d, mice were immunized with a mixture of vaccine and a recombinant soluble form of CR2 termed (CR2)2-IgG1 (Fig. 1). The (CR2)2-IgG1 construct consists of the first two short consensus repeats of murine CR2, which contain the C3d binding site, fused to the heavy chains of a mouse IgG1 molecule (7). Mice immunized with PPS14-C3d or a mixture of PPS14-C3d and a control murine IgG1 preparation had identical anti-PPS14 antibody responses. Coimmunization with PPS14-C3d and (CR2)2-IgG1 resulted in an ~75% reduction in serum anti-PPS14 IgM (1,767 ng/ml versus 7,256 ng/ml) at 10 days after primary immunization and a 94% decrease in IgG anti-PPS14 concentrations (13 ng/ml versus 214 ng/ml). Doubling the (CR2)2-IgG1 dose decreased anti-PPS14 IgM concentrations (1,262 ng/ml) to 17% of control values (7,256 ng/ml) and anti-PPS14 IgG concentrations (4 ng/ml) to 2% of control levels (214 ng/ml). These results demonstrate that interactions between C3d and CR2 are critical for the primary antibody response to PPS14-C3d.

We previously have shown that complement depletion at the time of primary immunization with either unmodified PPS14 or PPS14-OVA results in a marked enhancement of the secondary anti-PPS14 antibody response (21). In the present experiments, we limited our (CR2)2-IgG1 treatments to the time of primary immunization to determine whether inhibition of C3d binding to CR2 would influence the response to a second injection of PPS14-C3d. At the 0.5-µg dose of PPS14-C3d used in these experiments, control mice did not exhibit a boost in serum anti-PPS14 after a second similar injection of PPS14-C3d. In contrast, mice receiving (CR2)2-IgG1 at the time of primary immunization developed a strong secondary anti-PPS14 antibody response upon reimmunization with PPS14-C3d given without the inhibitor. At 10 days following secondary immunization, anti-PPS14 IgM concentrations in mice receiving (CR2)2-IgG1 were approximately five to six times those seen in control mice, while anti-PPS14 IgG concentrations were three times the values in control mice (Fig. 1). These results suggest that cross-linking C3d with antigen receptor at the time of primary immunization can have a negative effect on the secondary antibody response after reimmunization.

Effect of complement depletion on the anti-PPS14 antibody response to PPS14-C3d. If the effects of endogenous complement activation on the antibody response to PPS14 result primarily from binding of C3d(g) to the polysaccharide, then direct conjugation of C3d to PPS14 should eliminate the effects of complement depletion on the anti-PPS14 antibody response. Indeed, we found no significant differences in either the IgM or IgG anti-PPS14 antibody responses in mice immunized with or without CVF treatment at the time of primary immunization (Fig. 2). In this experiment, we found that at a dose of 0.5 µg, a third injection of PPS14-C3d was required to achieve an increase in anti-PPS14 concentrations above post-primary values. The decreased anti-PPS14 concentrations in CVF-treated versus control mice that were especially apparent after the third injection of PPS14-C3d were due to the presence of two very high responders in the control group. Thus, differences for the groups as a whole did not achieve statistical significance and disappeared altogether if these two mice were eliminated from the analysis (data not shown). These data confirm that C3d-CR2 interactions are paramount in complement-mediated effects on the antibody response to PPS14.

Anti-PPS14 antibody response after immunization with PPS14-C3d conjugates having high and low C3d/PPS14 ratios. Because the PPS14-C3d conjugate used in the (CR2)2-IgG1 experiment shown in Fig. 1 had a relatively high molar ratio (6.4:1) of C3d to PPS14, we wondered whether decreasing the amount of C3d incorporated into PPS14-C3d conjugates would result in an increased secondary anti-PPS14 antibody response. Thus, we compared the antibody response after immunization of mice with PPS14-C3d conjugates having low (2.2:1 and 3.0:1) and high (6.3:1) molar ratios of C3d to PPS14 (Fig. 3). We used a PPS14 dose of 1 µg rather than 0.5 µg to increase the likelihood that a boost in anti-PPS14 Ig would be induced by secondary immunization. Serum anti-PPS14 concentrations after primary immunization were similar in the three different treatment groups. After secondary immunization, maximum anti-PPS14 IgM concentrations occurred at 10 days postimmunization and were approximately twice as high in mice receiving the two lower-ratio conjugates as in those receiving the high-ratio conjugate. The serum anti-PPS14 IgM geometric mean concentrations (GMCs) were 15,872 ng/ml for mice receiving the 2.2:1-ratio PPS14-C3d conjugate, 17,477 ng/ml for mice receiving the 3.0:1-ratio conjugate, and 8,980 ng/ml for mice immunized with the 6.3:1-ratio conjugate. When concentrations at 10 days after secondary immunization were compared with those at 10 days after primary immunization, anti-PPS14 IgM levels for the 2.2:1-ratio group were 2.6 times post-primary concentrations, 6.2 times post-primary values for the 3.0:1 ratio group, but only 1.3 times post-primary concentrations for the 6.3:1 ratio group.

Differences were even more apparent for anti-PPS14 IgG concentrations, which were maximal at 25 days after secondary immunization. The serum anti-PPS14 IgG GMCs were 4,684 ng/ml for mice immunized with the 2.2:1-ratio PPS14-C3d conjugate, 1,688 ng/ml for mice receiving the 3.0:1-ratio conjugate, and 991 ng/ml for mice receiving the 6.3:1-ratio conjugate. When concentrations at 25 days after secondary immunization were compared with those at 25 days after primary immunization, anti-PPS14 IgG was boosted to 12.4 times post-primary concentrations for the 2.2:1-ratio group, to 15.3 times post-primary values for the 3.0:1-ratio group, and to 2.4 times post-primary concentrations for the 6.3:1-ratio group. Overall, it is apparent that the amount of C3d incorporated into PPS14-C3d conjugates can impact the magnitude of the secondary anti-PPS14 antibody response, with lower C3d incorporation having a favorable effect on the secondary response without impairing the primary antibody response.

Comparison of the anti-PPS14 antibody response to PPS14-C3d and PPS14-OVA in CBA/N xid mice. Differences in the responses of nude mice (20) and CVF-treated mice (21) to PPS14-C3d versus PPS14-OVA support the notion that PPS14-C3d retains the characteristics of a TI-2 antigen, while PPS14-OVA behaves more like a TD antigen. We sought further evidence to distinguish the two types of PPS14 conjugate by performing immunizations in CBA/N xid mice, which lack expression of Bruton's tyrosine kinase (Btk). The lack of an antibody response in CBA/N xid mice has historically been described as a defining characteristic of TI-2 antigens (14). When we immunized mice with either PPS14-C3d or PPS14-OVA, the anti-PPS14 antibody response was significantly impaired in CBA/N xid mice compared with CBA/J control mice. At 10 days after secondary immunization, GMC anti-PPS14 IgM was 5,071 ng/ml in CBA/J mice immunized with PPS14-C3d but only 8 ng/ml in CBA/N xid mice (P = 5 x 10–9). The corresponding anti-PPS14 IgG concentrations were 454 ng/ml in CBA/J mice and 5 ng/ml in CBA/N xid mice (P = 3 x 10–7). For mice immunized with PPS14-OVA, the anti-PPS14 IgM GMC was 21,860 ng/ml in CBA/J mice at 10 days post-secondary immunization but only 84 ng/ml in CBA/N xid mice (P = 6 x 10–8). The corresponding anti-PPS14 IgG concentrations for these mice were 56,646 ng/ml in CBA/J mice and 18 ng/ml in CBA/N xid mice (P = 2 x 10–7). Thus, the anti-PPS14 antibody response to both PPS14-C3d and PPS14-OVA conjugates requires functional Btk.

CBA/N xid mice were then given a third injection of the conjugate vaccines to see if differences in the antibody responses to PPS14-C3d and PPS14-OVA would become apparent. These data are shown in Fig. 4. Twenty-five days after a third injection of PPS14-C3d, CBA/N xid mice failed to increase serum anti-PPS14 IgM above preimmune levels (GMC, 2 ng/ml post-tertiary immunization versus 6 ng/ml pre-primary immunization) and anti-PPS14 IgG concentrations also did not increase (GMC, 3 ng/ml post-tertiary versus 8 ng/ml pre-primary). In contrast, mice immunized with PPS14-OVA had significant increases in anti-PPS14 IgM (621 ng/ml post-tertiary versus 5 ng/ml preimmune, P = 0.002) and anti-PPS14 IgG (2,238 ng/ml post-tertiary versus 8 ng/ml preimmune, P = 0.00003). Anti-PPS14 antibody concentrations were also significantly greater for mice immunized with PPS14-OVA compared with those immunized with PPS14-C3d (Fig. 4). Taken together, the data from this experiment suggest that both PPS14-C3d and PPS14-OVA behave as TI-2 antigens with respect to a requirement for Btk, but that PPS14-OVA conjugates are significantly less dependent on Btk than are PPS14-C3d conjugates.


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DISCUSSION
 
The majority of C3d vaccine constructs reported to date follow the principles outlined in the original publication on the adjuvant effect of C3d, in which Dempsey et al. engineered DNA constructs containing monomeric, dimeric, or trimeric C3d coupled end to end with a molecule of hen egg lysozyme (1). When dimers or trimers of C3d were incorporated, the individual C3d molecules were separated by 14-amino-acid spacers containing multiple copies of glycine and serine (1). To our knowledge, our PPS14-C3d conjugate vaccines are the only C3d conjugates examined thus far that do not consist of C3d conjugated to a protein. In contrast to TD protein antigens, PPS14 behaves as a TI-2 antigen, so we have been careful to use conjugation chemistry that preserves the TI-2 nature of the PPS14 (20, 21). By activating the PPS14 with the organic cyanylating reagent 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (10) and using highly purified murine plasma C3d, we are able to generate direct conjugates of C3d and PPS14 without the use of any chemical cross-linkers or amino acid spacers, both of which could act as TD carriers. Thus, the adjuvant effects of C3d in our PPS14-C3d conjugates should be mediated by interactions between C3d and CR2 (and/or CR1). However, the recent report of the ability of C3d in protein conjugates to enhance the protein-specific antibody response in CD21/35–/– mice (6) provided the impetus for the studies reported here, in which we specifically examined the roles of C3d-CR2 interactions and endogenous complement activation in the humoral immune response to PPS14-C3d.

The primary antibody response to PPS14-C3d was significantly inhibited by simultaneous injection of mice with (CR2)2-IgG1, a soluble form of CR2 that prevents the binding of C3d to its receptors on B cells and follicular dendritic cells. Thus, binding to CR2 is required for the enhancing effect of PPS14-C3d on the anti-PPS14 antibody response. Further, the lack of effect of CVF treatment on the antibody response to PPS14-C3d suggests that the effects of complement activation on the antibody response to PPS14 are mediated primarily via interactions between C3d(g) and CR1/2. How then do we reconcile our results with those demonstrating an ability of C3d conjugate vaccines to enhance anti-protein antibody responses in CD21/CD35–/– mice (6)? Besides obvious differences in the antigens used, differences in the C3d preparations used in the conjugate vaccines may provide one explanation. Whereas we used C3d purified from mouse plasma, CD21/CD35–/– mice (6) were immunized with either biotinylated human C3dg tetramers (9) conjugated to streptavidin or with recombinant human immunodeficiency virus gp120 fused with linear multimers of murine C3d containing 10-amino-acid linkers between the C3d molecules (3). Either the human C3dg or the amino acid linkers could behave as TD carriers in mice. However, even if the C3d preparations used in the studies involving CD21/CD35–/– mice were behaving as TD carriers, it is unclear whether conjugation of a TD protein carrier to a different TD antigen has any effect on the antibody response to the latter antigen. Another potential explanation for our results is that differences in the immune systems of CD21/CD35–/– mice and wild-type mice could affect the response to C3d conjugates. For example, CD21/CD35–/– mice have significant alterations in splenic B-cell subpopulations compared with wild-type mice (5). Third, it is possible that binding of (CR2)2-IgG1 could prevent C3d from binding to ligands or receptors other than CR1 and CR2, assuming that the binding site on C3d for the ligand were in close proximity to that for CR2. This sort of interference would not occur in CD21/35–/– mice. For example, two C3d binding proteins distinct from lymphocyte CR1 and CR2 have been identified on mouse platelets (17), but it is unknown whether binding of C3d to these receptors has any immunomodulatory effects. Binding sites have also been demonstrated on human factor H for C3d, but the putative factor H-binding region on C3d is remote from the CR2 binding site (8). At our current level of understanding of mouse C3d, we believe it remains most likely that the effects of (CR2)2-IgG1 on the immune response to PPS14-C3d are mediated by interference with C3d binding to CR1 and CR2.

Inhibition of C3d-CR2 interactions at the time of primary immunization by treatment of mice with (CR2)2-IgG1 resulted in an enhanced anti-PPS14 antibody response to a second injection of PPS14-C3d, a result similar to the enhanced secondary response seen in mice treated with CVF at the time of primary immunization with either unmodified PPS14 or PPS14-OVA (21). Therefore, it is likely that the effects of CVF treatment in mice immunized with PPS14 and PPS14-OVA were a consequence of the absence of C3d(g) deposition on PPS14, which prevented C3d-CR2 interactions at the time of antigen receptor engagement. The results of experiments showing an enhanced secondary anti-PPS14 response after immunization with PPS14-C3d conjugates having low C3d/PPS14 ratios provide additional evidence that C3d-CR2 interactions are important in regulating the magnitude of the secondary antibody response. A recent report showed that the antigen-specific memory B-cell response was essentially normal after immunization of CD21/CD35–/– mice with two different viral antigens (2), suggesting that complement activation can have different effects on primary and memory (secondary) antibody responses. CD21 costimulation was shown to be necessary for induction of Blimp-1 and XBP-1, transcriptional regulators that drive the differentiation of B cells into plasma cells (2). Our results suggest that inhibition of C3d-CR2 interactions at the time of primary immunization not only can suppress plasma cell differentiation, but also can lead to enhanced development of memory B cells.

Finally, our results provide additional evidence that the C3d in our PPS14-C3d conjugate vaccines is not behaving as a TD protein carrier. Treatment of mice with CVF at the time of primary immunization with PPS14-C3d was without significant effect on either the primary or secondary anti-PPS14 antibody response. In contrast, CVF treatment at the time of primary immunization with PPS14 conjugated to the TD protein carrier OVA resulted in marked inhibition of the primary anti-PPS14 antibody response and significant enhancement of the secondary response (21). If C3d in PPS14-C3d conjugates were simply behaving as a TD carrier, the effects of CVF treatment should have been identical to those observed in mice immunized with PPS14-OVA. In addition, CBA/N xid mice immunized with PPS14-C3d failed to mount an antibody response even after three injections with vaccine, consistent with the TI-2 nature of the PPS14 antigen. Although CBA/N xid mice immunized with PPS14-OVA also had a significantly impaired anti-PPS14 antibody response compared with CBA/J control mice, the response was significantly greater than that seen after immunization with PPS14-C3d. The results of PPS14-OVA immunization in CBA/N xid mice are consistent with previously reported results describing the effects of immunization of CBA/N xid mice with conjugates of PPS14 and bovine serum albumin (22). Coupled with our previously reported results showing a marked impairment of the primary anti-PPS14 antibody response in BALB/c nu/nu mice immunized with PPS14-OVA but not PPS14-C3d (20) and differences in the degree of isotype switching (20) and the magnitude of secondary antibody responses to the two different conjugates, these data suggest that PPS14-C3d retains the characteristics of a TI-2 antigen.

In summary, the effects of purified murine plasma C3d conjugated to a TI-2 polysaccharide antigen are mediated by binding to CR1/2 and do not require the activation of endogenous complement. C3d-CR2 interactions at the time of primary immunization can also modulate the antibody response to a subsequent injection of conjugate vaccine. Finally, conjugation of C3d to pneumococcal capsular polysaccharide does not appear to confer the properties of a TD antigen on the polysaccharide; instead it retains the properties of a TI-2 antigen. These results do not necessarily apply to all antigens, nor to all C3d preparations, and suggest that the mechanisms of C3d effect may need to be examined on a conjugate-by-conjugate basis.


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ACKNOWLEDGMENTS
 
We thank Alexander H. Lucas (Children's Hospital Oakland Research Institute) for providing monoclonal antibodies to PPS14.

This work was supported by National Institutes of Health grant AI-49250.


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FOOTNOTES
 
* Corresponding author. Mailing address: Children's Hospital Oakland Research Institute, 5700 Martin Luther King, Jr. Way, Oakland, CA 94609-1673. Phone: (510) 450-7630. Fax: (510) 450-7910. E-mail: stest{at}chori.org. Back

Editor: J. N. Weiser


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Infection and Immunity, November 2005, p. 7311-7316, Vol. 73, No. 11
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.11.7311-7316.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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