production-e-coli-expressed-self-assembling-protein-nanoparticles-for

Production of <em>E. coli</em>-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

Current influenza vaccines should be improved by the addition of universal influenza vaccine antigens in order to protect against multiple virus strains. We used our self-assembling protein nanoparticles (SAPNs) to display the two conserved influenza antigens M2e and Helix C in their native oligomerization states. To further improve the immunogenicity of the SAPNs, we designed and incorporated the TLR5 agonist flagellin into the SAPNs to generate self-adjuvanted SAPNs. We demonstrate that addition of flagellin does not affect the ability of SAPNs to self-assemble and that they are able to stimulate TLR5 in a dose-dependent manner. Chickens vaccinated with the self-adjuvanted SAPNs induce significantly higher levels of antibodies than those with unadjuvanted SAPNs and show higher cross-neutralizing activity compared to a commercial inactivated virus vaccine. Upon immunization with self-adjuvanted SAPNs, mice were completely protected against a lethal challenge. Thus, we have generated a self-adjuvanted SAPN with a great potential as a universal influenza vaccine.

Vaccination with self-adjuvanted protein nanoparticles provides protection against lethal influenza challenge.

We have developed FMP014, a vaccine candidate against Plasmodium falciparum malaria, which is comprised of 60 identical monomer protein chains that form an icosahedral shaped self-assembling protein nanoparticle (SAPN). Each monomer contains selected P. falciparum Circumsporozoite Protein (PfCSP) CD4+ and CD8+ epitopes, universal T epitopes, portions of the α-TSR domain, and 6 repeats of the NANP motifs of the PfCSP. Here we describe the conditions that are required for successful scale-up and cGMP manufacturing of FMP014 with a yield of ≈1.5g of drug substance per 100g of wet bacterial paste. When adjuvanted with an Army Liposomal Formulation (ALF) based adjuvant, the nanoparticle vaccine is highly immunogenic and prevents infection of mice by an otherwise lethal dose of transgenic P. berghei sporozoites expressing the full-length PfCSP.

Development of a self-assembling protein nanoparticle vaccine targeting Plasmodium falciparum Circumsporozoite Protein delivered in three Army Liposome Formulation adjuvants.

Self-assembling protein nanoparticles: a promising tool against lethal influenza?

The parasitic disease malaria remains a major global public health concern and no truly effective vaccine exists. One approach to the development of a malaria vaccine is to target the asexual blood stage that results in clinical symptoms. Most attempts have failed. New antigens such as P27A and P27 have emerged as potential new vaccine candidates. Multiple studies have demonstrated that antigens are more immunogenic and are better correlated with protection when presented on particulate delivery systems. One such particulate delivery system is the self-assembling protein nanoparticle (SAPN) that relies on coiled-coil domains of proteins to form stable nanoparticles. In the past we have used de novo designed amino acid domains to drive the formation of the coiled-coil scaffolds which present the antigenic epitopes on the particle surface.

The use of a P. falciparum specific coiled-coil domain to construct a self-assembling protein nanoparticle vaccine to prevent malaria.

We designed and produced a self-assembling protein nanoparticle. This self-assembling protein nanoparticle contains five CD8 HLA-A03-11 supertypes-restricted epitopes from antigens expressed during 's lifecycle, the universal CD4 T cell epitope PADRE, and flagellin as a scaffold and TLR5 agonist. These CD8 T cell epitopes were separated by N/KAAA spacers and optimized for proteasomal cleavage. Self-assembling protein nanoparticle adjuvanted with TLR4 ligand-emulsion GLA-SE were evaluated for their efficacy in inducing IFN-γ responses and protection of HLA-A*1101 transgenic mice against . Immunization, using self-assembling protein nanoparticle-GLA-SE, activated CD8 T cells to produce IFN-γ. Self-assembling protein nanoparticle-GLA-SE also protected HLA-A*1101 transgenic mice against subsequent challenge with Type II parasites. Hence, combining CD8 T cell-eliciting peptides and PADRE into a multi-epitope protein that forms a nanoparticle, administered with GLA-SE, leads to efficient presentation by major histocompatibility complex Class I and II molecules. Furthermore, these results suggest that activation of TLR4 and TLR5 could be useful for development of vaccines that elicit T cells to prevent toxoplasmosis in humans.

Protein nanovaccine confers robust immunity against .

Infectious bronchitis virus (IBV) affects poultry respiratory, renal and reproductive systems. Currently the efficacy of available live attenuated or killed vaccines against IBV has been challenged. We designed a novel IBV vaccine alternative using a highly innovative platform called Self-Assembling Protein Nanoparticle (SAPN). In this vaccine, B cell epitopes derived from the second heptad repeat (HR2) region of IBV spike proteins were repetitively presented in its native trimeric conformation. In addition, flagellin was co-displayed in the SAPN to achieve a self-adjuvanted effect. Three groups of chickens were immunized at four weeks of age with the vaccine prototype, IBV-Flagellin-SAPN, a negative-control construct Flagellin-SAPN or a buffer control. The immunized chickens were challenged with 5x10(4.7) EID50 IBV M41 strain. High antibody responses were detected in chickens immunized with IBV-Flagellin-SAPN. In ex vivo proliferation tests, peripheral mononuclear cells (PBMCs) derived from IBV-Flagellin-SAPN immunized chickens had a significantly higher stimulation index than that of PBMCs from chickens receiving Flagellin-SAPN. Chickens immunized with IBV-Flagellin-SAPN had a significant reduction of tracheal virus shedding and lesser tracheal lesion scores than did negative control chickens. The data demonstrated that the IBV-Flagellin-SAPN holds promise as a vaccine for IBV.

Article	Year
Vaccination with self-adjuvanted protein nanoparticles provides protection against lethal influenza challenge. Nanomedicine : nanotechnology, biology, and medicine\| PubMed ID: 27593488	2017
Development of a self-assembling protein nanoparticle vaccine targeting Plasmodium falciparum Circumsporozoite Protein delivered in three Army Liposome Formulation adjuvants. Vaccine\| PubMed ID: 28274638	2017
Self-assembling protein nanoparticles: a promising tool against lethal influenza? Nanomedicine (London, England)\| PubMed ID: 28621581	2017
The use of a P. falciparum specific coiled-coil domain to construct a self-assembling protein nanoparticle vaccine to prevent malaria. Journal of nanobiotechnology\| PubMed ID: 28877692	2017
Protein nanovaccine confers robust immunity against . NPJ vaccines\| PubMed ID: 29263879	2017
A self-adjuvanted nanoparticle based vaccine against infectious bronchitis virus. PloS one\| PubMed ID: 30216376	2018

Peter Burkhard

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