( A ) ADDomer design. Three evolutionary nonconserved segments (boxed) in the protomer were engineered by BioBrick design into exchangeable cassettes. One cassette (yellow) is located within the VL, while two cassettes, RGD1 (light green) and RGD2 (dark green), are located within the loop containing a functional RGD tripeptide sequence. Numbers indicated amino acid boundaries of BioBrick cassettes as well as VL and RGD loop. ( B ) Left: A 3.5-Å cryo-EM map of the ADDomer particle formed by 60 protomers. The rigid core is colored blue, and more flexible regions comprising the loops are colored cyan and green. Right: The corresponding atomic model is shown in gray. One penton (center) is highlighted, with individual protomers colored red, orange, green, cyan, and blue, respectively. ( C ) Side view of a penton formed by five protomers. Flexible VL and RGD loop are drawn in dashed lines. ( D ) Closeup view of the RGD loop and VL in ADDomer. Residues 156 to 170 in VL and 314 to 366 in RGD loop in the BioBrick format maintain their flexibility, essential for functionalization by epitope insertion. ( E ) Superimposition of the fiber-binding region in the ADDomer cryo-EM structure (cyan) with apo Ad3 [Protein Data Bank (PDB) ID: 4AQQ; marine blue] and fiber peptide–bound Ad3 (PDB ID: 4AR2; magenta) crystal coordinates. The fiber peptide (PDB ID: 4AR2) is drawn in a ball-and-stick representation. ( F ) Individual ADDomer protomer is shown in a side view (left) with a putative metal-binding cluster boxed in between the crown (top) and jellyroll fold (bottom) domains. Zoomed-in views of the boxed region (right) depict the atomic model in the EM density contoured at two different levels, σ = 4 (blue) and σ = 10.5 (red). Four juxtaposed sulfurs (yellow spheres) likely coordinate a metal ion. ( G ) Left: Central channel of an ADDomer penton. Right: Ca 2+ coordination as seen previously in the Ad3 penton base protein crystal structure (PDB ID: 4AR2) is not observed. The helices fencing the central channel are rearranged, resulting in a different conformation of E466. ( H ) Interface between two pentons (A1 and A2) in the ADDomer (cyan). The zoomed-in view depicts the boxed region with the corresponding Ad3 crystal structure (magenta) superimposed. Domain swapping is not observed in the ADDomer. Instead, this interface is stabilized by mutual hydrogen bonds between S61 side chains and E59 peptide backbones from neighboring protomers (right).

Comparison of primary sequences of penton-forming protomers from many adenovirus serotypes revealed particularly two regions of high variability in length and sequence, called variable loop (VL) and arginine-glycine-aspartic acid (RGD) loop. For instance, VL comprises 20 amino acids in Ad3 while only 9 amino acids in Ad41. RGD loop plasticity is even more pronounced in Ad12, spanning 11 residues, in contrast to 74 residues in Ad2. The VL has been shown previously to accommodate an influenza-derived immunogenic eptitope ( 20 ). The RGD loop contains a conserved tripeptide motif (-RGD-) mediating integrin-based internalization into target cells ( 21 ). We reasoned that these regions, VL and RGD loop, given their polymorphism, would be ideally suited to accommodate one or several antigenic epitopes each. Therefore, we redesigned the protomer-encoding gene adopting a “BioBrick” format ( 22 ) to facilitate multiple epitope insertions. We inspected the crystal structure of Ad3 dodecahedron ( 17 ) and identified suitable loci adjacent to secondary structure motifs flanking the loops. The RGD loop in wild-type Ad3 is considerably more extended than the VL. As the tripeptide is important for cell internalization, we decided to split the RGD loop into two sections before and after the RGD motif, which we kept unaltered. We inserted unique restriction sites to create three independent loci for epitope display, one in the VL and two in the RGD loop to facilitate insertion of synthetic DNAs encoding for antigenic epitopes of choice in the resulting plug-and-play multiepitope display platform, the ADDomer ( Fig. 1A and figs. S1 and S2). We expressed ADDomer using MultiBac, the baculovirus/insect cell system we developed for complex protein biologics ( 23 ), resulting in highly purified ADDomer (fig. S1) with excellent culture yields (50 mg from a 50-ml culture) and negligible nucleic acid or endotoxin contamination.

ADDomer structure and mechanism

Unlocking ADDomer to future structure-based design, we determined its molecular architecture by high-resolution cryo–electron microscopy (cryo-EM) (Fig. 1, B to H, and figs. S3 and S4). For parts of the process, we used public cloud resources, implementing image processing and refinement software for this purpose. Our cryo-EM structure, at up to 3.2-Å resolution, reveals ADDomer adopting the familiar dodecahedron, formed by 12 pentons in a quasi-spherical arrangement, with the RGD loop and VL densely decorating the particle surface (Fig. 1, B and C, fig. S3, and table S1). Our structure shows that the plasticity of the RGD loop and VL, essential for antigenic epitope presentation, is maintained in ADDomer (Fig. 1D).

Crystal structures of wild-type Ad3 dodecahedron with and without fiber peptide bound exist at lower resolution (3.8 and 4.8 Å, respectively) (17). Close inspection of our cryo-EM structure reveals notable differences. The fiber-binding cleft adopts a rearranged conformation, seemingly in between the geometries observed in the crystals of apo- and fiber-bound Ad3 dodecahedron (Fig. 1E) (17). The protomer represents a two-domain architecture, with a crown region comprising the VL and RGD loop abutting a jellyroll fold mediating multimerization (Fig. 1, D and F). In our cryo-EM structure, we observed additional density in between the crown and jellyroll domains, juxtaposed to four sulfur-containing amino acids (M131, M138, M458, and C539), consistent with a tetradentate coordination by a metal ion, possibly zinc or iron (Fig. 1F). We posit that a structural metal ion at this interdomain interface could be important for stabilizing this protein fold.

In the Ad3 dodecahedron crystal, a Ca2+ ion occupied a central cavity lined by glutamates (E466) in α-helices from different protomers (Fig. 1G). No corresponding density is observed by cryo-EM. The Ad3 crystallization conditions contained CaCl 2 , whereas we did not supplement Ca2+ during ADDomer purification to occupy this site. Consequently, glutamates E466 do not extend into the cavity, but the corresponding α-helices are slightly shifted in the ADDomer.

A hallmark in the Ad3 crystal structure was strand-swapping between extended N-terminal regions of neighboring protomers, deemed essential to dodecahedron structural integrity (17). Unexpectedly, we did not observe this strand-swapping by cryo-EM (Fig. 1H and fig. S4C). Rather, in ADDomer, two neighboring protomers engage in hydrogen bonds in the vicinity of the twofold axis, extending from the S61 side chain to the peptide backbone of E59 and vice versa. We can exclude that the differences observed are due to ADDomer amino acid additions and substitutions within the VL and RGD loop, as they are in the crown domain and thus sterically too distant to have a bearing on the N-terminal domain, or the formation of the hitherto unobserved metal binding site, respectively.

Strand-swapping and Ca2+ ions would conceivably stabilize the dodecahedron. We did not observe either of these features by cryo-EM and thus wondered about ramifications for ADDomer stability. Thermal shift assays evidenced a melting temperature of 54°C for ADDomer and no noticeable melting below 45°C (fig. S5A). We challenged ADDomer integrity by storing for weeks at room temperature, freezing and thawing, and incubating at 45°C. Negative-stain EM revealed uniformly stable ADDomer (fig. S5, B to E). Thus, we can conclude that ADDomer maintains highly advantageous thermotolerance, begging the question what exactly the source of this remarkable stability may be.

ADDomer (30 nm) is within the size range (20 to 200 nm) of particles readily drained to lymph nodes, potentiating uptake by antigen-presenting cells and cross-presentation (5, 6). We analyzed ADDomer uptake by human cells, including monocytes and monocyte-derived dendritic cells (MDDCs), confirming efficient internalization (Fig. 2, A and B). We next investigated lymph node distribution in mice injected with fluorescently labeled ADDomer (Fig. 2, C and D). Rapid draining to the right inguinal lymph node was observed irrespective of the mode of administration. No signal was found in the opposite left inguinal node serving as internal control. Mesenteric and axillary nodes evidenced rapid but not lasting signal (Fig. 2D). Our results underscore ADDomer capacity to drain rapidly to the nearest lymph node and to efficiently penetrate human cells, including antigen-presenting lymphocytes.