Autologous albumin enhances the humoral immune response to capsular polysaccharide covalently coattached to bacteria-sized latex beads

Abstract

Abundant autologous proteins, like serum albumin, should be immunologically inert. However, individuals with no apparent predisposition to autoimmune disease can develop immune responses to autologous therapeutic proteins. Protein aggregation is a potential major trigger of these responses. Adsorption of proteins to particles provides macromolecular size and may generate structural changes in the protein, resembling aggregation. Using aldehyde/sulfate latex beads coated with murine serum albumin (MSA), we found that BALB/c mice mounted MSA-specific IgG responses that were dependent on CD4(+) T cells. IgGs were specific for MSA adsorbed to solid surfaces and noncross-reactive with human, bovine, or pig albumins. T cells induced in response to MSA augmented the primary and induced boosted secondary IgG and IgM responses specific for the T cell-independent antigen, capsular polysaccharide of Streptococcus pneumoniae type 14 (PPS14), when the latter was attached to the same bead. Similar to the anti-MSA IgG response, the boosted PPS14-specific IgG secondary response was CD4(+) T-cell dependent, displayed a typical carrier effect, and was enhanced by, but did not require, Toll-like receptor stimulation. These results provide a potential mechanism for the induction of responses to autoantigens unable to induce specific T-cell responses, and provide new insights into polysaccharide-specific immunity.

Keywords: Autoimmunity; Autoreactive memory responses; Humoral responses; Immunity to bacteria.

Figure 1. PPS14 and autologous MSA co-attached to latex beads induce PPS14-specific antibody responses in a T cell-dependent manner

(A) BALB/c and BALB/c athymic nude mice (n=7), were immunized on days 0 and 14 (arrows) with ≈ 2×10⁸ latex beads, coated with PPS14 combined with glycine or MSA, in alum+CpG-ODN. Each dose of the corresponding beads was adjusted to contain approximately 230ng of serologically active PPS14. (B) Female BALB/c mice (n=5) were injected with 1mg of anti-CD4 mAb (GK1.5) or polyclonal rat IgG2b (control), 32h before injection with 1µg of free PPS14, or 2.2 × 10⁸ PPS14+[MSA]-beads containing 80ng of PPS14, both admixed only with CpG-ODN. Mice received a secondary immunization 14 days later with the same antigen in the absence of anti-CD4 mAb or polyclonal rat IgG. Anti-MSA IgM responses are only shown for Panel A. Panel A and B shown different experiments using different bead preparations. Beads were always washed in PBS prior to immunization, to remove any antigen leaked free to the supernatant. Serum titers of IgG and IgM specific for PPS14 and MSA were determined by ELISA. Values are expressed as geometric mean ± SEM. *p < 0.05 (BALB/c versus athymic nude mice or controls relative to GK1.5 treated mice), #p< 0.05 (titer⁻¹ relative to the previous bleeding of each mouse group); Student's t-test.

Figure 2. Secondary PPS14-specific IgG responses to PPS14+[MSA]-beads are highly enriched in the expression of the 44.1-Idiotype

BALB/c mouse sera (n=21) collected 7–14 days after a primary (primary response) or 7 days after a secondary (secondary response) immunization at day 14 with latex beads coated with the antigen combinations indicated, were analyzed for the expression of the 44.1-Id in PPS14-specific IgG, IgG1 and IgM by inhibition ELISA. Sera were collected in three independent experiments. Mice received the primary immunization at day 0 and challenged at day 14. *p< 0.05 (primary relative to secondary response), #p< 0.05 (each group relative to the responses to PPS14+[PspA]-beads); Student's t-test.

Figure 3. Average avidity of PPS14-specific IgG responses to PPS14+[MSA]-beads

The average avidity (AI) of PPS14-specific IgG in serum was estimated by elution with sodium thiocyanate (NaSCN). Mice were immunized at days 0 and 14 with ≈2×10⁸ latex beads containing co-attached 230 ng of PPS14 and 202 ng PspA (PPS14+[PspA]) or with 42 ng MSA (PPS14+[MSA]) or in the absence of proteins, blocked with an excess of Gly (PPS14+[Gly]). All beads were admixed with CpG-ODN (without alum added). Sera tested (n=15–17) were from three independent experiments. Data show the avidity index (AI), as the molar concentration of NaSCN eluting 50% of the total content of PPS14-specific IgG in the serum sample. *p < 0.05 (titer⁻¹ relative to serum samples from the previous time point of each mouse group), #p < 0.05 (each group relative to the responses to PPS14+[PspA]-beads); Student's t-test.

Figure 4. MSA-specific IgG and PPS14-specific IgG secondary responses to PPS14+[MSA]-beads are induced only by MSA and/or PPS14 attached to either micro- or nanobeads; enhancement of responses by CpG-ODN, but not alum

Serum titers of PPS14-specific IgG in BALB/c mice (n=5) immunized at day 0 and 14 with (A) ≈2.2×10⁸ latex beads containing 280ng of PPS14 attached to beads coated with MSA (PPS14+[MSA]) or normal mouse sera (PPS14+[NMS]), previously collected from the same mice receiving the beads. (B) 2×10⁸ latex beads coated with MSA ([MSA]-beads), 30 ng of free PPS14, or a mixture of 2×10⁸ [MSA]-beads and 30ng of free PPS14 ([MSA]-beads + free PPS14). (C) ≈2×10⁸ PPS14+[MSA]-beads of a preparation in which the PPS14 attachment to the bead was not stabilized by reduction with cyanoborohydride and contained 209ng of PPS14 attached and 20ng of free PPS14, after >1year of storage. This preparation was used for immunization after washings with PBS that reduced the free PPS14 content to <0.8 ng (not stabilized, washed), or directly without washings (not stabilized, free PPS14), and compared to a preparation in which the Schiff’s bases were reduced with cyanoborohydride (stabilized, washed). (D) 2×10⁸ 0.96 µm in diameter, or 1.46 × 10¹¹ 0.15µm in diameter, PPS14+[MSA]-beads containing 200 ng of attached PPS14 (E) 3.5×10⁸ 0.96µm in diameter, or 4.5×10⁹ 0.15 µm in diameter, PPS14+[MSA]-beads containing 28 ng of attached PPS14. #p <0.05 (for C and D, primary [day 14] relative to secondary [day 21] responses); Student's t-test. (F) Anti-MSA IgG in sera of BALB/c mice (n=7) immunized at day 0 and 14 with 2×10⁸ beads coated with 100 ng MSA ([MSA]-beads) or glycine ([Gly]-beads) combined with 25µg CpG-ODN, or 50µg of purified MSA (free MSA) admixed with 25µg CpG-ODN and 13µg of alum. A mixture of CpG-ODN and alum (PBS) was used as control. (G) Serum titers of MSA-specific IgG and PPS14-specific IgG and IgM from BALB/c mice (n=7) immunized at day 0 and 14, with 2×10⁸ PPS14+[MSA]-beads containing 240ng of PPS14 attached to the beads. Beads were admixed for 1 hour at room temperature with 13µg of alum and 25µg CpG-ODN (alum+CpG-ODN), 25µg CpG-ODN alone (CpG-ODN), 13µg alum alone (alum), or in the same final volume of PBS (PBS).*p<0.05 (every group relative to mice immunized with PPS14+[MSA]-beads admixed with alum+CpG-ODN). Latex beads used in all the experiments, except for panel D and E, were 0.96µm in diameter. One of three (panel G), or two (panels A, D and F) independent experiments is shown.

Figure 5. Sera from mice immunized with latex beads coated with MSA contain IgG that specifically recognized MSA adsorbed to solid surfaces

(A) Inhibitory capacity of soluble and particulate forms of MSA. Mouse sera collected 7 days after a secondary immunization with 2×10⁸ PPS14+[MSA]-beads were incubated overnight at 4°C with 100µg/well of soluble MSA, soluble BSA or 41ng/well MSA attached to beads, alone ([MSA]-beads) or in combination with PPS14 (PPS14+[MSA]-beads), all of them diluted in 2%OVA. This is the highest number of beads that can be reliably used in the assay. Sera incubated with the same amount of latex beads coated only with Gly ([Gly]-beads) were used as negative control. 1/20,000 dilutions of biotinylated polyclonal goat anti-MSA was used as positive control for inhibition. Unbound anti-MSA IgG was detected by ELISA in wells coated with 5µg/ml of MSA, and blocked with 2% OVA. *p <0.05 (relative to group immunized with [Gly]-beads); Student's t-test. Three independent experiments carried out in duplicate. (B) Purified serum albumins from human (HSA), bovine (BSA) and mouse (MSA) of two different degrees of purification (95% or 99% purity) were resolved in 4–20% SDS-PAGE Tris-Glycine gradient gels under non-reducing conditions. The supernatant of a boiled sample of [MSA]-beads is also included. The separated proteins were stained with Coomassie Blue (protein stain), or transferred to 0.45µm nitrocellulose membranes and detected by incubation with 1/100 dilution of a pool of five sera collected before immunization (preimmune), or 7 days after a secondary immunization with PPS14+[MSA]-beads. Immune sera from two independent experiments (Exp1 and Exp 2), using different PPS14+[MSA]-bead preparations are shown. Bound antibodies were detected with polyclonal goat anti-mouse IgG conjugated to horseradish-peroxidase followed by 4-chloronaphtol. The location of mouse globulins in the blot, which reacts with the secondary goat anti-mouse IgG(H+L), is indicated by arrows.

Figure 6. PPS14-specific IgG responses to PPS14+[MSA]-beads are enhanced by TLR ligand adjuvants, but not alum

BALB/c mice (n=7) were immunized at day 0 and 14, with 2×10⁸ PPS14+[MSA]-beads containing 240ng of PPS14 attached to the beads. Beads were admixed for 1 hour at room temperature before immunization with 25µg CpG-ODN, 50µg Pam₃CSK₄ (Pam₃Cys) or 20µg synthetic monophosphoryl lipid A (MPL). No alum was added. (A) serum titers of MSA- and PPS14-specific IgG and IgM. (B) Serum titers of IgG isotypes during the primary (day 14) and secondary (day 21) responses. For serum titers of anti-MSA IgG, only secondary responses are shown because primary responses were mostly undetectable. One of two independent experiments is shown. *p < 0.05 (every group relative to mice immunized with PPS14+[MSA]-beads admixed with CpG-ODN). #p < 0.05 (primary [day 14] versus secondary [day 21] responses); Student's t-test.

Figure 7. Previous immunity to [MSA] beads, boosts the PPS14-specific IgG responses to PPS14+[MSA]-beads

BALB/c mice (n=7) were pre-immunized with 2×10⁸ beads coated with 37ng MSA ([MSA]-beads), 13 days before the start of the immunization schedule. Control mice received PBS (naïve). At day 0 and 14 (indicated by arrows), mice were immunized with 450ng of free PPS14 or ≈10⁸ latex beads coated with, PPS14 and MSA (PPS14+[MSA]-beads), containing 160ng of PPS14 and 42ng MSA attached to the bead. Beads and free PPS14 were admixed for 1 hour with 25µg CpG-ODN, before immunization. Free PPS14 also included 13µg of alum. *p < 0.05 (groups preimmunized with [MSA]-beads, relative to naïve mice); Student's t-test One of two independent experiments is shown.