Immunosilencing a Highly Immunogenic Protein Trimerization Domain

Background: Trimerization domains are commonly used to stabilize trimeric protein vaccines and therapeutics.

Results: The GCN4-based isoleucine zipper domain induces strong antibody responses in vivo but this can be overcome by introducing glycans.

Conclusion: Appropriately positioned glycans can effectively immunosilence the GCN4-based trimerization domain.

Significance: Immunosilencing trimerization domains could be important for the exploitation of trimerization domains in protein vaccines and therapeutics.

Abstract

Many therapeutic proteins and protein subunit vaccines contain heterologous trimerization domains, such as the widely used GCN4-based isoleucine zipper (IZ) and the T4 bacteriophage fibritin foldon (Fd) trimerization domains. We found that these domains induced potent anti-IZ or anti-Fd antibody responses in animals when fused to an HIV-1 envelope glycoprotein (Env) immunogen. To dampen IZ-induced responses, we constructed an IZ domain containing four N-linked glycans (IZN4) to shield the underlying protein surface. When fused to two different vaccine antigens, HIV-1 Env and influenza hemagglutinin (HA), IZN4 strongly reduced the antibody responses against the IZ, but did not affect the antibody titers against Env or HA. Silencing of immunogenic multimerization domains with glycans might be relevant for therapeutic proteins and protein vaccines.

Introduction

Several small protein domains can facilitate trimerization of proteins. The two most widely used trimerization domains in biochemical and biomedical research are the isoleucine zipper (IZ)³ based on the GCN4 transcriptional activator from Saccharomyces cerevisae, and the foldon domain of the bacteriophage T4 fibritin protein (Fd) (1, 2). IZ consists of α-helices in a coiled-coil heptad repeat, in which the first (a) and fourth (d) amino acid residues in each heptad repeat determine the oligomerization state of the protein (3). Isoleucines at the a and d positions facilitate trimerization while alternating isoleucine/leucine or leucine/isoleucine can confer dimerization or tetramerization, respectively, which can be exploited for engineering dimeric, trimeric, or tetrameric proteins (1, 3, 4). In contrast, Fd consists of three β-hairpins, which assemble into a β-propeller-like structure (5). The Fd trimer is stabilized by hydrogen-bonding, hydrophobic interactions, and salt-bridges between each (2, 5).

IZ and Fd are commonly fused to soluble proteins that depend on trimerization for their therapeutic activity or proper antigenic and immunogenic structure. These include cancer therapeutics that have been tested in clinical trials, such as the TNF superfamily member CD40 ligand (6–8), as well as therapeutics that have been tested preclinically, such as OX40 ligand (9) and TRAIL (10). Experimental protein vaccines, some, which are considered for clinical trials, also exploit IZ and/or Fd and include the spike proteins of human immunodeficiency virus (HIV-1) (11–20), respiratory syncytial virus (21, 22), and influenza virus (4, 23–28).

Despite their extensive use in preclinical studies, the immunogenicity of the yeast-derived IZ and bacteriophage-derived Fd has not been properly evaluated. When IZ and Fd would be routinely used for future clinical applications and vaccines, immune responses against IZ, and Fd could affect the effectiveness of these therapeutics and vaccines. For example, with repeated administration of therapeutic proteins, IZ- or Fd-specific antibodies (Abs) might enhance systemic clearance of the protein thereby decreasing the efficiency of the therapy (29, 30).

Here we report that the IZ and Fd trimerization domains induce potent Ab responses in vaccinated animals. To dampen the anti-IZ response, we designed an IZ variant with four potential N-linked glycosylation sites (PNGS) at strategic positions in the heptad repeat domain of IZ (IZN4). Protein trimerization was not affected by the addition of four glycans to the IZ domain. Moreover, IZN4 induced significantly lower IZ-specific Ab responses in vaccinated animals when fused to two different antigens, without hampering the immune response against these antigens. This novel IZN4 trimerization domain should be useful for trimeric protein vaccines and therapeutics.

EXPERIMENTAL PROCEDURES

Plasmids and Mutagenesis

The expression vector for the HIV-1 subtype B JRFL Env SOSIP.R6 (Env) with a C-terminal IZ (RMKQIEDKIEEILSKIYHIENEIARIKKLIGER) and histidine tag has been described elsewhere extensively (13). In short, the Env construct used in this study (Env-IZ) contains a disulfide between gp120 and gp41; an improved furin cleavage site (R6); an I559P mutations to improve trimerization; a codon-optimized GCN4-pII isoleucine zipper (IZ); a GGGGTGGGGTG-linker between the IZ and Env moiety; a C-terminal histidine tag (His-tag) (1, 13, 31, 32). The codon-optimized influenza hemagglutinin (HA) sequence (GenBank^TM: ACU65077.1, residues 10–509) was synthesized (Mr. Gene) and was cloned to replace the Env sequence in the same expression vector yielding [HA]GGGGTGGGGTGRMKQI EDKIEEILSKIYHIENEIARIKKLIGERHHHHHHHH (HA-IZ). To obtain Env-Fd and HA-Fd, the codon-optimized T4 bacteriophage Fd sequence (GYIPEAPRDGQAYVRKDGEWVLLSTFL) was cloned in place of the IZ sequence. Env-IZ variants with the different PNGS were generated using the Quickchange mutagenesis kit (Agilent). For HA-IZN4 we replaced the IZ in HA-IZ with the IZN4 (NGTGRMKQIEDKIENITSKIY NITNEIARIKKLIGNRT) sequence from Env-IZN4. For the constructs used in rabbit vaccinations (i.e. Env-IZ, Env-IZN4, HA-IZ, and Env-IZN4), a stop-codon was introduced before the His-tag to avoid the induction of antibodies against the tag.

Reagents

mAbs were gifts or purchased from the following sources: Polymun Scientific (2G12); Progenics Pharmaceuticals (PA1, soluble CD4, CD4-IgG2); J. Mascola and P. Kwong, Vaccine Research Center (VRC01); D. Burton, The Scripps Research Institute (PGT121); J. Robinson, Tulane University (48d); H. Katinger, through the AIDS Research and Reference Reagent Program (2F5); Sino Biologicals (H5 HA mAb); Genscript (anti-His-tag mAb).

Cell Culture and Transient Expression

HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin (100 units/ml), and streptomycin (100 μg/ml). Cells were transfected with PEImax (Polysciences) and plasmid DNA expressing IZ- and Fd-fusion proteins. For the experiments shown in Fig. 3A, plasmid DNA encoding the protease furin was co-transfected in a 1:1 ratio with Env-IZ or Env-IZN4 plasmid to induce cleavage of the gp120 and gp41_ecto-IZN4 subunits of the Env-IZN4 proteins. When indicated, kifunensine (5 μg/ml, Cayman Chemical) was added during transfection to block mannose trimming of N-linked glycans (33). To remove oligomannose glycans from Env-IZ and Env-IZN4, 20 μl supernatant of kifunensine-treated HEK293T cells was treated with 1 μl of endoglycosidase H (Sigma Aldrich) according to the manufacturer's instructions.

FIGURE 3.

IZN4 is fully glycosylated and preserves its trimerization capacity. A, 293T cells were transfected with Env-IZ and Env-IZN4 in the presence of excess furin to force Env cleavage to facilitate the analysis of the gp41_ecto-IZ and gp41_ecto-IZN4 domains by SDS-PAGE. Proteins were blotted using an anti-His-tag Ab. The transfections were performed in the absence (lanes 1 and 2) and presence (lanes 3–6) of kifunensine to block synthesis of complex glycans (33). The kifunensine-treated 293T cell supernatants were subsequently treated with EndoH to deglycosylate the protein (lanes 3 and 4). The ladder between lanes 5 and 6 marks all possible nine glycoforms of gp41_ecto-IZ and gp41_ecto-IZN4 that could be readily resolved in kifunensine produced proteins. B, BN-PAGE analysis of Env and different Env-IZ variants, followed by blotting with 2G12. Monomeric, dimeric, trimeric Env forms, and aggregates are indicated by arrows.

SDS-PAGE, BN-PAGE, and Western Blotting

Proteins in HEK293T cell supernatants were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blue native PAGE (BN-PAGE). Western blotting was performed using anti-His-tag mAb (0.13 μg/ml), PA1 (0.2 μg/ml), or anti-H5 HA mAb (0.5 μg/ml) for SDS-PAGE and 2G12 (0.1 μg/ml) for BN-PAGE. Proteins were detected using a horseradish peroxidase (HRP)-labeled goat anti-mouse Ab (for anti-His-tag, PA1, and anti-H5 HA mAbs) or goat anti-human Ab (for 2G12).

Anti-IZ, -IZN4, and -Fd ELISA

HEK293T cell supernatants containing Env-IZ, Env-IZN2/3a/3b/4, HA-IZ, HA-IZN4, or HA-Fd were diluted 1:2 in TBS/10%FCS and immobilized on Ni-NTA HisSorb 96-well plates (Qiagen) (100 μl/well) for 2 h at room temperature. After three washing steps with Tris-buffered saline (TBS), sera from mice, rats, and rabbits was serially diluted in 2% skimmed milk/20% sheep serum and added for 2 h at room temperature. Subsequently, HRP-labeled goat-anti-mouse, goat-anti-rat, or goat-anti-rabbit Abs (Jackson Immunoresearch) were added for 1 h at a 1:5000 dilution (final concentration 0.33 μg/ml) in TBS/2% skimmed milk, followed by five washes with TBS/0.05% Tween-20. Colorimetric detection was performed using a solution containing 1% 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich), 0.01% H₂O₂, 100 mm sodium acetate and 100 mm citric acid. Midpoint titers were calculated using GraphPad Prism (version 5.01).

Anti-Env and Anti-HA ELISA

Anti-gp120 Ab titers were measured by D7324-capture ELISA as described previously (34). Anti-HA Ab titers were measured using HA-Fd immobilized on Ni-NTA HisSorb 96-well plates. Reactivity of Env proteins with mAbs or receptor mimics was measured by anti-Env-IZ ELISA immobilized on Ni-NTA plates as described above, using HRP-labeled goat-anti-human as the secondary Ab.

Competition ELISA

HEK293T supernatant containing D7324-tagged Env-IZ (35) was diluted 1:2 in TBS/10% FCS and coated on D7324-coated ELISA plates for 2 h at room temperature. HA-Fd, HA-IZ, Env-Fd containing transfection supernatants were concentrated 8-fold using Vivaspin20 filters (Sartorius) and incubated with rabbit sera for 1h at RT. Subsequently, sera were serially diluted in 2% skimmed milk with 20% sheep serum, supplemented with the corresponding 8-fold concentrated supernatant. Subsequent ELISA assay steps were performed as described above. Relative Ab binding to the Env and IZ domains in rabbit serum was assessed using the midpoint binding titers as follows (Fig. 1C): For Env: ((Env-Fd − HA-Fd)/(HA-IZ + Env-Fd)) × 100%; for IZ: ((HA-IZ − HA-Fd)/(HA-IZ + Env-Fd − HA-Fd)) × 100%.

FIGURE 1.

Two widely used trimerization domains are immunogenic in animals. Midpoint ELISA titers from sera of Env-IZ vaccinated (A) rabbits (week 4), rats (week 4), and mice (week 6) or (B) Env-Fd vaccinated rabbits (week 22) against IZ and Fd. Horizontal lines indicate the median midpoint titer. C, pie charts for individual rabbit sera showing the relative Env- and IZ-specific responses in a competition ELISA for the rabbit sera used in A (see “Experimental Procedures” for calculations).

Immunizations

For the experiments shown in Fig. 1A four outbred NMRI mice, four Wistar rats, and four New Zealand White rabbits were immunized in the abdominal dermis with endotoxin-free Env-IZ plasmid DNA using gene gun technology at week 0, 2, and 4. For mice and rats we used 20 μg of plasmid DNA and 125 μg of DNA was used for rabbits. For the experiment shown in Fig. 1B, sera from rabbits immunized with Env-Fd (Env from clade B isolate YU2 with C-terminal foldon domain, described extensively elsewhere) (36) were obtained from an ongoing vaccination experiment in New Zealand White rabbits that is done in collaboration with IAVI. These rabbits were immunized with 30 μg of Env-Fd protein in ISCOMATRIX^TM adjuvant at weeks 0, 4, and 20. In previous vaccination experiments using similar protocols, five to six animals per group provided enough statistical power to detect significant differences between two groups in binding antibody measurements (35, 37). Therefore, we used six New Zealand White rabbits for the experiments in Fig. 5, A and B that were immunized in the abdominal dermis at week 0, 2, 4, 8 with 125 μg endotoxin-free DNA encoding Env-IZ, Env-IZN4, HA-IZ, and Env-IZN4 using gene gun technology(35, 37). All protocols dealing with animal manipulations were in accordance with guidelines published by FELASA (Federation of European Animal Science Association) and GV-SOLAS (German Society of Laboratory Animal Science) and were reviewed by the Harlan, MFD Diagnostics, or Davids Biotechnologie Animal Care Committees, as appropriate.

FIGURE 5.

IZN4 is effectively immunosilenced without compromising antigen-specific responses. Rabbits were immunized on week 0, 2, 4, and 8. Midpoint binding titers of week 10 sera against IZ and IZN4 of rabbits that were immunized with (A) Env-IZ and Env-IZN4; (B) HA-IZ and HA-IZN4. C, gp120 binding titers and (D) HA binding titers of the sera used in A and B, respectively. Statistical analysis was performed using a Mann-Whitney test. *: p ≤ 0.05; **: p ≤ 0.01.

RESULTS

Two Widely Used Protein Trimerization Domains Are Highly Immunogenic

To determine the immunogenicity of the IZ domain, we immunized mice, rats and rabbits with plasmid DNA encoding the HIV-1 Env from strain JRFL containing an IZ domain at the C terminus (Env-IZ) (32, 35). In addition, the immunogenicity of the Fd domain was studied in rabbits that were immunized in an independent study with Env from the strain YU2, containing the Fd domain at the C terminus (Env-Fd). Sera from all immunized animals were tested for IZ- and Fd-specific Ab responses by testing the response against fusion proteins in which IZ and Fd were fused to influenza hemagglutinin from strain A/Vietnam/1194/2004 (HA). We observed high levels of Abs against IZ in sera from Env-IZ-vaccinated mice, rats and rabbits (Fig. 1A). Similarly, we observed strong anti-Fd response in sera from Env-Fd-vaccinated rabbits (Fig. 1B), but not vice versa. Thus, both IZ and Fd are highly immunogenic. To analyze the relative amounts of IZ-specific versus Env-specific Abs, we performed a competition ELISA in which we depleted either Env- or IZ-specific Abs. In the four tested rabbits, 36 to 61% of the relative binding Ab response against the Env-IZ immunogen was specific for the IZ-domain (Fig. 1C). We conclude that the relatively small IZ trimerization domain (12 kDa) was highly immunogenic in rabbits in comparison with Env (∼400 kDa).

Design of an Immunosilenced IZ Trimerization Domain: IZN4

Silencing of unwanted immunodominant Ab epitopes can be achieved by using N-linked glycans (N-glycans) (28, 38, 39). Therefore we investigated whether it would be possible to shield the immunogenic IZ protein surface with N-glycans. The IZ domain is a heptad repeat in which isoleucines on the first (a) and fourth (d) position in the α-helix direct trimerization (Fig. 2A). Introduction of PNGS by means of the amino acid motif aspargine-X-serine/threonine (NX(S/T)) is only possible on the cde positions in the heptad repeat, because other options would eliminate the isoleucines responsible for oligomerization, or would result in a steric clash of the N-glycan with a neighboring α-helix. We chose to use NXT motifs over NXS, because NXT motifs have a higher probability of glycosylation (40). Based on these considerations we selected four positions for insertion of NXT motifs that were ultimately combined in a variant that had all four motifs (Fig. 2, B and D). Note that the first PNGS is in the linker region between Env and IZ. In silico prediction suggested that introducing four NXT motifs in IZ (IZN4) had a negligible effect on the tertiary structure of the IZ (Fig. 2B). Modeling of IZ glycans on the IZN4 trimer showed that the accessibility of underlying protein surface was dramatically reduced (Fig. 2C).

FIGURE 2.

Design of an immunosilenced GCN4-based trimerization domain. A, helical wheel presentation of the coiled coil heptad repeat of IZ. The a and d positions in gray contain isoleucines and are important for trimerization. The predicted glycosylation sites at position c contain an asparagine (N) (solid circle) and a threonine (T) at position e (dotted circle). B, the predicted three-dimensional structure (predicted with iTasser (50)) of the three subunits of IZN4 (yellow) was overlaid with the original IZ structure (in light green, PDB: 1GCM). The numbers indicate the glycosylation sites at the c positions in IZN4. C, model of the fully glycosylated structure of IZN4. The predicted structure from B was used to model complex glycans on IZN4 using GlyProt with default settings (51). The glycans are represented in pink spheres. D, alignment of the amino acid sequences of the different glycosylated IZ constructs. The gp41_ecto domain of Env and the linker are also indicated. Numbers above the sequences correspond to the numbers in B.

IZN4 Is Glycosylated and Trimerizes Efficiently

We sequentially introduced the NXT motifs at positions 1, 2, 3 and 4 of IZ in the context of Env-IZ (Fig. 2D): positions 1 and 4 (IZN2); positions 1, 2, and 4 (IZN3a); positions 1, 3, and 4 (IZN3b); positions 1, 2, 3, and 4 (IZN4). To verify that the newly introduced PNGS were glycosylated, we tested the glycan occupancy on IZ and IZN4 by separating gp41_ecto-IZ and gp41_ecto-IZN4 from gp120 by furin cleavage and subsequent reducing SDS-PAGE (31). gp41_ecto-IZN4 migrated slower than gp41_ecto-IZ in SDS-PAGE (Fig. 3A, lanes 1 and 2). To confirm that the slower migration of gp41_ecto-IZN4 was due to N-glycans, we produced Env-IZ and Env-IZN4 in the presence of furin and the mannose-analog kifunensine, resulting in exclusively uniform oligomannose Man_8/9GlcNAc₂ glycans (33). The supernatant was then treated with Endoglycosidase H (EndoH), which removes oligomannose N-glycans. Deglycosylated gp41_ecto-IZ and gp41_ecto-IZN4 showed the same fast migrating band (Fig. 3A, lanes 3 and 4) indicating that the size difference in lane 1 and 2 was caused by glycans. The number of occupied PNGS in gp41_ecto-IZ or gp41_ecto-IZN4 was determined by reducing SDS-PAGE of the kifunensine produced furin-cleaved Env-IZ and Env-IZN4 proteins in which each glycoform of gp41_ecto-IZ and gp41_ecto-IZN4 was readily resolved (Fig. 3A, lanes 5 and 6). Gp41_ecto-IZ contains four PNGS in gp41_ecto, but only one to three PNGS were occupied by an N-glycan (Fig. 3A, lane 5), consistent with earlier findings on gp41 glycan occupancy (41). In contrast, gp41_ecto-IZN4 contains eight PNGS and we found occupancy of five to eight N-glycans (Fig. 3A, lane 6), suggesting that all four PNGS per α-helix in the IZN4 were occupied, showing that we successfully added a total of twelve N-glycans to the IZN4 trimer.

The Env, Env-IZ, Env-IZN2/3a/3b/4 constructs were then expressed in a mammalian cell-line (HEK293T) and analyzed by native PAGE analysis. The addition of up to four N-glycans to IZ had no negative effect on the trimerization propensity of Env-IZ, but might slightly increase the trimer proportion in the supernatant. In contrast, removing the IZ trimerization domain resulted in formation of Env monomers, dimers, trimers, and aggregates (Fig. 3B) (13). We conclude that the introduction of four PNGS did not disrupt IZ trimerization, but allowed the attachment of four N-glycans per IZ α-helix.

Antigenic Epitopes Are Shielded by Glycans in IZN4

To examine whether the introduced N-glycans can shield immunogenic epitopes of the IZ domain, we tested sera of Env-IZ vaccinated rabbits for binding to Env-IZ or Env-IZ-glycan variants in ELISA. Introduction of N-glycans at the N and C terminus of the IZ domain (Env-IZN2) decreased binding and the binding was further reduced by the addition of N-glycans on position 2 (Env-IZN3a). Env-IZN3b and Env-IZN4 showed inefficient binding to the sera of Env-IZ vaccinated rabbits (Fig. 4A). The remaining binding can be attributed to Env-directed Abs.

FIGURE 4.

Glycosylation of IZ decreases binding of Env-IZ sera and preserves antigenicity of Env-IZ. A, sera (week 12) of four Env-IZ vaccinated rabbits, described previously (35), were tested for their ability to recognize Env-IZ, Env-IZN2, Env-IZN3a, Env-IZN3b, and Env-IZN4 in ELISA. The bars represent the midpoint binding titers relative to that of Env-IZ. Statistical analyses were performed using the Kruskal-Wallis test followed by Dunn's Multiple Comparison test. *: p ≤ 0.05; **: p ≤ 0.01. B, representative ELISA curves of Env-IZ and Env-IZN4 with representative Abs against four different epitopes on Env: V3 glycans (2G12 and PGT121), MPER (2F5), CD4 binding site (soluble CD4 and VRC01), and the CD4 induced state (48 d).

To verify that glycosylation of the IZ domain did not affect the folding of the Env antigen, we compared the antigenic structure of Env-IZ and Env-IZN4 using antibodies that bind to four distinct antigenic sites on Env: the CD4 binding site (VRC01), the CD4-induced epitopes (48d), the membrane-proximal external region (MPER), located adjacent to the IZ domain (2F5), and the glycan-dependent supersite centered around N332 (2G12 and PGT121) (Fig. 4B). Binding of these antibodies was similar for both Env-IZ and Env-IZN4, indicating that the antigenic structure and glycosylation of Env was unaltered by glycosylation of IZ.

IZN4 Is Immunosilenced but Preserves Antigen-specific Responses

To investigate whether IZ glycosylation resulted in decreased anti-IZ Ab responses, a vaccination experiment was conducted using Env and influenza HA as the model antigens. Rabbits were immunized with Env-IZ, Env-IZN4, HA-IZ, or HA-IZN4. After 10 weeks we determined the Ab binding titers to IZ and IZN4 for all four vaccine groups using the reciprocal proteins, i.e. the anti-IZ and anti-IZN4 responses for the Env-IZ and Env-IZN4 vaccinated rabbits were measured by using immobilized HA-IZ and HA-IZN4 and vice versa. Animals vaccinated with Env-IZ and HA-IZ raised 3–4-fold higher IZ-specific Ab titers compared with animals vaccinated with Env-IZN4 and HA-IZN4, independent of the fused antigen (Fig. 5, A and B). Moreover, IZN4-vaccinated rabbits did not induce a higher IZN4-specific Ab response, showing that the addition of N-linked glycans to the IZ decreases its overall immunogenicity without shifting to an IZN4-specific response (Fig. 5, A and B). The decreased immunogenicity of IZN4 did not alter the Ab response against the Env and HA immunogens. The gp120 binding titers for Env-IZ and Env-IZN4 vaccinated animals were similar (Fig. 5C). HA-IZN4 vaccinated animals induced slightly higher levels of HA Ab compared with HA-IZ, but this was not statistically significant (Fig. 5D). The responses against gp120 were at least 20-fold lower than those against HA, consistent with observations that the Env is a relatively poor immunogen (42). Collectively, these results show that IZN4 was effectively immunosilenced, without compromising the antigen-specific response.

DISCUSSION

Heterologous trimerization domains are used in many (pre)clinical studies, but an extensive analysis of their immunogenicity has never been reported. We found that two of the most widely used trimerization domains, IZ and Fd, induced potent Ab responses. This is relevant for a number of therapeutic proteins as well as protein subunit vaccines that utilize these domains (4, 6–27, 43).

An important question is whether an immune response against a trimerization domain affects efficacy of the therapeutic protein or vaccine, and/or might be harmful. Several key issues need to be considered. First, Abs against IZ or Fd might facilitate immune complex formation, which may result in an adjuvant effect and could actually be beneficial for vaccines (44). For therapeutic (self) proteins the formation of immune complexes could break tolerance to the endogenous protein and result in an autoimmune response. Immune complex formation might also alter clearance rates of the therapeutic proteins and vaccines thereby affecting their efficacy (30, 45). Second, the introduction of immunodominant epitopes on a trimerization domain could potentially act as a decoy for more relevant epitopes on the vaccine antigen (46). Third, when trimerization domains would become more widely used in humans, pre-existing immunity against such domains might decrease the efficacy of therapeutic proteins and vaccines.

To negate any potential adverse effects of immunogenic trimerization domains, we designed a novel IZ-based trimerization domain where immunodominant epitopes are shielded by glycans (IZN4). It might also be possible to use this strategy to immunosilence the Fd domain, but the β-propeller-like structure of Fd might be less amendable to the addition of N-glycans. IZN4 was occupied by twelve N-linked glycans per trimer, trimerized efficiently and did not impact the antigenic structure of a model Env antigen. When IZN4 was fused to two different antigens (Env and HA) and used in immunization experiments, the levels of IZ-directed Abs were considerably reduced. We also considered that IZ glycosylation might impact the response against the attached antigen. Some studies have shown that silencing of an immunodominant epitope by adding N-glycans can redirect the immune response to other antigenic regions (28, 38), but other studies failed to show such an effect (39, 47). In our study, the decrease in Ab response against the immunodominant IZ domain did not significantly increase the Env- or HA-specific response.

Although the IZN4 response was 4-fold lower than IZ, IZN4-directed Abs were still detected (Fig. 5, A and B). This indicates that IZN4 is weakly immunogenic, possibly because of the high number of charged hydrophilic amino acids at the C terminus of IZN4 (48). However, we cannot completely rule out that the residual binding comes from Abs binding to the linker regions between Env/HA and IZ, that were similar in the constructs used for immunization and detection. To further reduce the immunogenicity of IZN4 one could attach more than four N-glycans to IZ, although such a high density of PNGS might hamper efficient glycan attachment to all sites and/or affect the trimerization propensity. Alternatively, one could remove B cell epitopes by replacing the charged residues on the surface of IZ by neutral amino acids (48). Furthermore, one could predict and silence immunodominant T cell epitopes on IZ to dampen T cell help (49).

In conclusion, we found that commonly used protein trimerization domains can be highly immunogenic, but they can be immunosilenced by the addition of N-glycans. The immunosilenced IZN4 domain might be a useful tool for protein vaccines and therapeutics.