Aluminum hydroxide influences not only the extent but also the fine specificity and functional activity of antibody responses to tick-borne encephalitis virus in mice

Abstract

Aluminum hydroxide is the most widely used adjuvant in human vaccines and serves as a potent enhancer of antibody production. Its stimulatory effect strongly depends on the adsorption of the antigen to the adjuvant, which may influence antigen presentation and, as a consequence, the fine specificity of antibody responses. Such variations can have functional consequences and can modulate the effectiveness of humoral immunity. Therefore, we investigated the influence of aluminum hydroxide on the fine specificity of antibody responses in a model study in mice using an inactivated purified virus particle, the flavivirus tick-borne encephalitis (TBE) virus, as an immunogen. To dissect and quantify the specificities of polyclonal antibodies in postimmunization sera, we established a platform of immunoassays using recombinant forms of the major target of neutralizing antibodies (protein E) as well as individual domains of E (DIII and the combination of DI and DII [DI+DII]). Our analyses revealed a higher proportion of neutralizing than virion binding (as detected by enzyme-linked immunosorbent assay) antibodies after immunization with aluminum hydroxide. Furthermore, the induction of antibodies to DIII, a known target of potently neutralizing antibodies, as well as their contributions to virus neutralization were significantly greater in mice immunized with adjuvant and correlated with a higher avidity of these antibodies. Thus, our data provide evidence that aluminum hydroxide can lead to functionally relevant modulations of antibody fine specificities in addition to its known overall immune enhancement effect.

Fig 1

Structure of flaviviruses and recombinant antigens used for serological analyses. (A) Schematic model of the flavivirus virion. The capsid, which is surrounded by a lipid membrane, contains the viral RNA genome and multiple copies of the capsid protein C. Proteins E and M are anchored in the viral membrane and form an icosahedral lattice. A soluble form of an E protein (sE) dimer lacking the stem and anchor regions is indicated. (B) Ribbon diagram of the soluble E protein monomer of TBE virus (Protein Data Bank [PDB] code 1SVB; side view) (22). In DIII, the positions of amino acid replacements used to generate the so-called DIII lateral ridge mutant are indicated and highlighted in green. (C) Schematic representations of recombinant proteins used for serological analyses. Color code (A to C): protein M, pink; protein E, DI, red; DII; yellow; DIII, blue; anchor, gray; stem, purple; capsid protein, gray; RNA, dark gray; lipid membrane, yellow; His tag, black; Strep tag, orange; TR (thioredoxin), brown; and DIII-lr mutations, green.

Fig 2

Quality controls of antigens used in the study. (A) Coomassie-stained SDS-PAGE (5% phosphate gel) (44) and Western blotting (B) with a polyclonal mouse serum specific for C and E proteins (lanes 1, purified live virus; lanes 2, formalin-inactivated virus used for immunization). Monomeric and formalin cross-linked oligomeric bands of C and E proteins are labeled. (C) Coomassie-stained SDS-PAGE (15% Laemmli gel) of recombinant proteins used in ELISA and depletion assays. Molecular mass standards (Std) are indicated on the left. (D to F) Blocking ELISAs with MAbs specific for DI (IC3), DII (A3), and DIII (B4) and recombinant antigens (sE, orange; DI+II, green; DIII, blue) compared to sE isolated from purified live virions (v-sE, gray) (22).

Fig 3

TBE virion-specific antibody response after immunization without and with aluminum hydroxide (−Alu and +Alu, respectively). Virion ELISA (A), NT (B), and fold difference of titers (C) obtained with the +Alu serum pool compared to the −Alu serum pool in virion ELISA and NT. (D) Virion ELISA avidities. The data represent the means from at least three independent experiments, and error bars indicate the standard errors of the means. n.s., not significant.

Fig 4

ELISA reactivities with virion and recombinant antigens of −Alu and +Alu serum pools. (A) ELISA titers against virion, soluble recombinant E (sE), DI+DII, and DIII of TBE virus and recombinant WN virus sE. (B) Fold difference of titers obtained with the +Alu serum pool compared to the −Alu serum pool in virion and recombinant protein ELISAs. The data represent the means from at least three independent experiments, and error bars indicate the standard errors of the means.

Fig 5

(A) Ribbon diagram of DIII (side view) with amino acid positions affecting the binding of DIII-specific MAbs (B1 to B4) highlighted by colored spheres: red, B1 and B4 (59, 72); yellow, B2 (46); orange, B3 (unpublished data). The newly generated DIII-lr mutant contains replacements at amino acid positions 309 and 333 (highlighted in green). (B to F) Titration curves of His tag-specific and TBE DIII-specific MAbs. MAb anti-His tag (B), MAb B1 (C), MAb B2 (D), MAb B3 (E), and MAb B4 (F) were used in ELISA using wild-type (wt; orange line) and DIII-lr mutant (mut; blue line) TBE-DIII-TR-His as antigens. The data represent the means from three independent experiments, and the error bars indicate the standard errors of the means.

Fig 6

Antibody response to the TBE DIII-lr epitope. (A) Reduced reactivity of −Alu and +Alu serum pools with the DIII-lr mutant, expressed as a percentage of the DIII wt ELISA reactivity. (B) Relative avidities of −Alu and +Alu postimmunization sera for the DIII-lr mutant. The data represent the means from at least three independent experiments, and error bars indicate the standard errors of the means.

Fig 7

ELISA and NT analyses of −Alu and +Alu serum pools after depletion with recombinant DIII. (A) Percent remaining reactivity after antibody depletion with DIII, as determined by DIII ELISA. (B) Percent remaining reactivity after antibody depletion with DIII as determined by virion ELISA. (C) Percent remaining reactivity after antibody depletion with DIII as determined by TBE virus neutralization assay based on the data presented in panels E (−Alu) and F (+Alu). (D) Relative avidities of DIII antibodies in the postimmunization sera determined by avidity ELISA using DIII as an antigen. (E and F) NT virus titration curves of −Alu (E) and +Alu (F) serum pools before (red solid lines) and after (red dotted lines) antibody depletion with DIII. Black and gray curves represent the virus control incubated without antibody and with a negative-control serum pool of 10 naive mice, respectively. The data represent the means from at least three independent experiments, and error bars represent the standard errors of the means.