In Vivo Assembly of Nanoparticles Achieved through Synergy of Structure‐Based Protein Engineering and Synthetic DNA Generates Enhanced Adaptive Immunity

Abstract

Nanotechnologies are considered to be of growing importance to the vaccine field. Through decoration of immunogens on multivalent nanoparticles, designed nanovaccines can elicit improved humoral immunity. However, significant practical and monetary challenges in large‐scale production of nanovaccines have impeded their widespread clinical translation. Here, an alternative approach is illustrated integrating computational protein modeling and adaptive electroporation‐mediated synthetic DNA delivery, thus enabling direct in vivo production of nanovaccines. DNA‐launched nanoparticles are demonstrated displaying an HIV immunogen spontaneously self‐assembled in vivo. DNA‐launched nanovaccines induce stronger humoral responses than their monomeric counterparts in both mice and guinea pigs, and uniquely elicit CD8+ effector T‐cell immunity as compared to recombinant protein nanovaccines. Improvements in vaccine responses recapitulate when DNA‐launched nanovaccines with alternative scaffolds and decorated antigen are designed and evaluated. Finally, evaluation of functional immune responses induced by DLnanovaccines demonstrates that, in comparison to control mice or mice immunized with DNA‐encoded hemagglutinin monomer, mice immunized with a DNA‐launched hemagglutinin nanoparticle vaccine fully survive a lethal influenza challenge, and have substantially lower viral load, weight loss, and influenza‐induced lung pathology. Additional study of these next‐generation in vivo‐produced nanovaccines may offer advantages for immunization against multiple disease targets.

Incorporation of a Novel CD4+ Helper Epitope Identified from Aquifex aeolicus Enhances Humoral Responses Induced by DNA and Protein Vaccinations

“…We verified the engineered variant DLnano_CD4MutLS_GT8 incorporating the LS3-KO epitope could still assemble homogenously by expressing this new construct in vitro and performing size exclusion chromatography (SEC) of the lectin-column-purified DLnano_CD4MutLS_GT8 transfection supernatant. SEC showed CD4MutLS_GT8 assembled homogenously into 60-mer (single peak observed on the SEC trace centering at 12.33 mL retention volume) similar to what we previously observed for the wildtype eOD-GT8-60mer ( Figure 3 B) ( Xu et al., 2020 ). Additionally, Size Exclusion Chromatography Multi Angle Light Scattering (SEC-MALs) analysis determined the molecular weight of CD4MutLS_GT8 to be around 2 MDa, close to the observed molecular weight of eOD-GT8-60mer ( Figure S2 A) ( Xu et al., 2020 ).…”

“…We previously observed that scaffold domains used to drive in vivo assembly of nanoparticle vaccines could sometimes induce CD4+ T cell responses ( Xu et al., 2020 ). Here, we compared CD4+ T cell responses elicited by various nanoparticle scaffolding domains, ferritin from Helicobacter pylori (3BVE), LS from Aquifex aeolicus , and the viral cage of Prototype Foamy Virus (PfV), in BALB/c immunized with DNA-launched GT8 nanoparticle vaccines that incorporate these respective protein domains (DLnano_3BVE_GT8, DLnano_LS_GT8, DLnano_PfV_GT8) ( Figure 1 A).…”

“…In our prior work, we used synthetic DNA delivery by electroporation to mediate in vivo assembly of nanoparticle vaccines and observed that some nanoparticle scaffolding domains (used to promote self-assembly of scaffolded antigens) could induce CD4+ T cell responses ( Xu et al., 2020 ). In this work, we examined and performed epitope mapping on several bacterial or viral scaffold protein domains and determined that lumazine synthase (LS) from Aquifex aeolicus contained very potent CD4-helper epitopes for both BALB/c and C57BL/6 mice.…”

“…In this work, we examined and performed epitope mapping on several bacterial or viral scaffold protein domains and determined that lumazine synthase (LS) from Aquifex aeolicus contained very potent CD4-helper epitopes for both BALB/c and C57BL/6 mice. LS can scaffold the assembly of 60 copies of HIV-priming antigen GT8, eOD-GT8-60mer, as well as other antigens ( Jardine et al., 2016 ; Xu et al., 2020 ). In silico binding analysis determined that the identified C57BL/6 CD4-helper epitope (LS-3) was predicted also to have high binding affinity (<100 nM) to several common human MHC-II alleles (HLA-DRB1∗07:01, HLA-DRB1∗15:01, and HLA-DRB5∗01:01) and binds to a complementary set of HLA-DR alleles as compared with PADRE.…”

Biomimetic Nanomaterial Strategies for Virus Targeting: Antiviral Therapies and Vaccines

“…In another demonstration, Xu et al reported the use of a DNA vaccine plasmid to induce the in vivo expression of a nanoparticle structural component comprising an HIV Env antigen fused with LS, and demonstrated its in vivo structural assembly in mice based on functional studies along with improved immune responses in mice and guinea pigs compared to immunization with a similar delivery strategy encoding antigen monomers only. [154] The design concept was further generalized to other natural protein scaffolds, such as ferritin nanoparticles, which also induced high immune responses and the delivery strategy could also be adapted to influenza HA antigen in order to endow mice with protective immunity against a lethal influenza virus challenge.…”