Although sample purity appeared to be lower than the dual-HIV and dual-flu cases, negative-stain EM confirmed the formation of combined flu/HIV iFerr as nonclustered well-formed particles (Figure 5a). electron PF-06424439 methanesulfonate microscopy and antibodyantigen binding analysis. Immunizations in guinea pigs with two-component ferritin particles, displaying diverse Env, HA, or both antigens, elicited neutralizing antibody responses against the respective viruses. The results provide proof-of-principle for the self-assembly of a two-component nanoparticle as a general technology for multimeric presentation of trimeric antigens. Keywords:antibody, HIV-1, immunogenicity, influenza, vaccine == Graphical Abstract == The presentation of viral antigens in a regular repetitive pattern on the surface of virus particles facilitates B cell activation.13Multimerization of antigens on engineered particles that mimic the geometric patterns observed for native viral proteins can lead to improved antibody responses.46Recently, ferritin, a self-assembling sphere-like nanoparticle consisting of 24 copies of a single protein, was used for the multimerization of influenza hemagglutinin (HA) antigens, resulting in the elicitation of antibodies with substantially improved neutralization breadth and potency in immunized animals.7The ferritin technology has since been extended to allow for the multimerization of other antigens such as the HIV-1 envelope PF-06424439 methanesulfonate (Env) glycoprotein8and EpsteinBarr virus gp350 glycoprotein9and can thus be viewed as a general platform for immunogen design. In addition to ferritin, other multimeric self-assembling platforms such as lumazine synthase have been assessed in immunogenicity studies,10and multicomponent nanoparticles have been developed through computational design11,12that may also be capable of multimeric antigen presentation.13 In the standard ferritinantigen formulation, an antigen is genetically fused to the N terminus of each of the 24 copies of the ferritin protein, allowing for the formation of outward-facing spike-like structures, which in the case of influenza PF-06424439 methanesulfonate HA assemble into eight trimer spikes (Figure 1a). The standard ferritin technology, however, can only permit the random coassembly of diverse antigens (by, e.g., coexpressing multiple ferritinantigen genes) and cannot guarantee the pattern nor the ratio of each antigen on a single particle. Here, we design a two-component ferritin, that allow the attachment of two different antigens in a regular geometric pattern and at an equal (1:1) ratio. We specifically tailor our designs for the presentation of trimeric antigens, which makes this technology especially applicable to viruses such as HIV-1, where antigens in a native-like trimer, rather than monomer, form are believed to be more optimal as immunogens.14These two-component ferritin particles allow for the presentation of four trimers, each for two distinct antigens. We show that two-component ferritin can form with two diverse HIV-1 Env antigens or two PF-06424439 methanesulfonate diverse influenza HA antigens, as well as both HIV-1 Env and influenza HA antigens displayed on a single two-component ferritin particle. == Figure 1. == Design of two-component ferritin Mouse monoclonal to ENO2 nanoparticles for attachment of diverse trimeric antigens. (a) Schematic of (upper) single-component ferritin (light blue) with eight copies of trimeric antigen A (black) and (lower) two-component ferritin (light blue and light green) with four copies each of trimeric antigens A (black) and B (gray). (b) Design of insect ferritin (left) heavy chain (HC, light blue) and PF-06424439 methanesulfonate (right) light chain (LC, light green) in monomer (upper) and particle (lower) form to allow attachment of trimeric antigens. To allow the presentation of two different antigens on the same particle, we used as a platform a ferritin molecule derived from the insectTrichoplusia ni(iFerr) since it self-assembles naturally as a 24-mer with 12 copies each of a heavy and light chain (we term these iFerr HC and iFerr LC, respectively) and an atomic-level structure of the iFerr particle was available, enabling structure-based design.15The location of the N-termini of the wild-type iFerr, however, was not optimal for attachment of trimeric antigens (Figure 1b). Hence, we modified the iFerr particle by deleting N-term residues from both iFerr HC and iFerr LC. This resulted in antigen attachment points on ferritin that formed an equilateral triangle with distances of 34 (HC) and 31 (LC) (Figure 1b), in line with the close to 30 distance between the C-term attachment points for influenza HA and HIV-1 Env. To determine whether these residue deletions would destabilize or affect the formation of ferritin particles, we performed negative-stain electron microscopy (EM). We observed spherical particles with a diameter of 145 11 , indicating that iFerr particles could successfully form even with N-terminal deletions in both chains (Figure 2a). == Figure 2. == Structural characterization of modified iFerr particles. (a) Negative-stain EM of designed insect ferritin particles with no antigen attached (scale bars indicate 50 and 15 nm (inset), respectively). (b) SDS-PAGE of iFerr particles with HIV-1 Env attached to iFerr heavy (HC) or light chains (LC), nonreduced (NR), or reduced (R). Lanes are as follows: M, molecular weight marker; lanes.