Escherichia coli as a New Platform for the Fast Production of Vault-like Nanoparticles: An Optimized Protocol.

Vaults are protein nanoparticles that are found in almost all eukaryotic cells but are absent in prokaryotic ones. Due to their properties (nanometric size, biodegradability, biocompatibility, and lack of immunogenicity), vaults show enormous potential as a bio-inspired, self-assembled drug-delivery system (DDS). Vault architecture is directed by self-assembly of the "major vault protein" (MVP), the main component of this nanoparticle. Recombinant expression (in different eukaryotic systems) of the MVP resulted in the formation of nanoparticles that were indistinguishable from native vaults. Nowadays, recombinant vaults for different applications are routinely produced in insect cells and purified by successive ultracentrifugations, which are both tedious and time-consuming strategies. To offer cost-efficient and faster protocols for nanoparticle production, we propose the production of vault-like nanoparticles in Escherichia coli cells, which are still one of the most widely used prokaryotic cell factories for recombinant protein production. The strategy proposed allowed for the spontaneous encapsulation of the engineered cargo protein within the self-assembled vault-like nanoparticles by simply mixing the clarified lysates of the producing cells. Combined with well-established affinity chromatography purification methods, our approach contains faster, cost-efficient procedures for biofabrication in a well-known microbial cell factory and the purification of "ready-to-use" loaded protein nanoparticles, thereby opening the way to faster and easier engineering and production of vault-based DDSs.

Self-Assembling Protein Nanoparticles in the Design of Vaccines: 2022 Update.

Vaccines constitute a pillar in the prevention of infectious diseases. The unprecedented emergence of novel immunization strategies due to the COVID-19 pandemic has again positioned vaccination as a pivotal measure to protect humankind and reduce the clinical impact and socioeconomic burden worldwide. Vaccination pursues the ultimate goal of eliciting a protective response in immunized individuals. To achieve this, immunogens must be efficiently delivered to prime the immune system and produce robust protection. Given their safety, immunogenicity, and flexibility to display varied and native epitopes, self-assembling protein nanoparticles represent one of the most promising immunogen delivery platforms. Currently marketed vaccines against the human papillomavirus, for instance, illustrate the potential of these nanoassemblies. This review is intended to provide novelties, since 2015, on the ground of vaccine design and self-assembling protein nanoparticles, as well as a comparison with the current emergence of mRNA-based vaccines.

All-in-one biofabrication and loading of recombinant vaults in human cells.

One of the most promising approaches in the drug delivery field is the use of naturally occurring self-assembling protein nanoparticles, such as virus-like particles, bacterial microcompartments or vault ribonucleoprotein particles as drug delivery systems (DDSs). Among them, eukaryotic vaults show a promising future due to their structural features,in vitrostability and non-immunogenicity. Recombinant vaults are routinely produced in insect cells and purified through several ultracentrifugations, both tedious and time-consuming processes. As an alternative, this work proposes a new approach and protocols for the production of recombinant vaults in human cells by transient gene expression of a His-tagged version of the major vault protein (MVP-H6), the development of new affinity-based purification processes for such recombinant vaults, and the all-in-one biofabrication and encapsulation of a cargo recombinant protein within such vaults by their co-expression in human cells. Protocols proposed here allow the easy and straightforward biofabrication and purification of engineered vaults loaded with virtually any INT-tagged cargo protein, in very short times, paving the way to faster and easier engineering and production of better and more efficient DDS.

Orientation of Antigen Display on Self-Assembling Protein Nanoparticles Influences Immunogenicity.

Self-assembling protein nanoparticles (SAPN) serve as a repetitive antigen delivery platform with high-density epitope display; however, antigen characteristics such as size and epitope presentation can influence the immunogenicity of the assembled particle and are aspects to consider for a rationally designed effective vaccine. Here, we characterize the folding and immunogenicity of heterogeneous antigen display by integrating (a) dual-stage antigen SAPN presenting the P. falciparum (Pf) merozoite surface protein 1 subunit, PfMSP119, and Pf cell-traversal protein for ookinetes and sporozoites, PfCelTOS, in addition to (b) a homogenous antigen SAPN displaying two copies of PfCelTOS. Mice and rabbits were utilized to evaluate antigen-specific humoral and cellular induction as well as functional antibodies via growth inhibition of the blood-stage parasite. We demonstrate that antigen orientation and folding influence the elicited immune response, and when appropriately designed, SAPN can serve as an adaptable platform for an effective multi-antigen display.

Nanovaccine based on self-assembling nonstructural protein 1 boosts antibody responses to Zika virus.

Self-assembling proteins may be generated after the addition of short specific amino acid sequences at both the N- and C-terminal ends. To date, this approach has not been evaluated regarding the impact of self-assembled proteins on the induction of immune responses. In the present study, we report the application of this experimental approach to the immunogenicity of protein antigens by measuring the antibody responses in mice immunized with nanoparticles made with a recombinant form of Zika virus nonstructural protein 1 (∆NS1). The results clearly indicated that ∆NS1-derived nanoparticles (NP-∆NS1) are assembled into a 3-dimensional structure with a high degree of multimerization. While ∆NS1 proved to be a weak immunogen, immunization with NP-∆NS1 enhanced subunit vaccines' immunogenicity with improved longevity in vaccinated mice. Thus, immunization with self-assembled antigens (nanovaccines) represents a new and promising strategy to enhance NS1-specific antibodies' induction based on purified recombinant proteins.

Nanoparticle Vaccines for Inducing HIV-1 Neutralizing Antibodies.

The enormous sequence diversity between human immunodeficiency virus type 1 (HIV-1) strains poses a major roadblock for generating a broadly protective vaccine. Many experimental HIV-1 vaccine efforts are therefore aimed at eliciting broadly neutralizing antibodies (bNAbs) that are capable of neutralizing the majority of circulating HIV-1 strains. The envelope glycoprotein (Env) trimer on the viral membrane is the sole target of bNAbs and the key component of vaccination approaches aimed at eliciting bNAbs. Multimeric presentation of Env on nanoparticles often plays a critical role in these strategies. Here, we will discuss the different aspects of nanoparticles in Env vaccination, including recent insights in immunological processes underlying their perceived advantages, the different nanoparticle platforms and the various immunogenicity studies that employed nanoparticles to improve (neutralizing) antibody responses against Env.