In this protein-in-profile, we demonstrate scalable cell-free expression of immunogenic hepatitis B core carrier VLP and the conjugation of antigens to preassembled VLPs for rapid adaptation to different pathogens. By leveraging this technology, vaccine developers can gain access to a flexible and efficient platform that accelerates the creation of new vaccines.
Vaccine Development Powered by VLPs
Virus-like particles (VLPs) are non-replicating, genome-free, multi-protein structures that mimic the structure of viruses. They are produced by expressing antigenic proteins that self-assemble into particles, which are then taken up by dendritic cells to activate the adaptive immune response. Their non-infectious nature, virus-like structure, and high immunogenicity make them a promising vaccine development platform.
VLPs have been utilized in several commercially available vaccines, such as those against hepatitis B virus (HBV) and human papillomavirus (HPV), and are notable for their ability to induce both innate and adaptive immune responses. The significance of VLPs lies in their potential to revolutionize vaccine development by offering a reliable and cost-effective platform that can be rapidly produced and tailored for various diseases.1,2
Vaccine Development Powered by VLPs
Virus-like particles (VLPs) are non-replicating, genome-free, multi-protein structures that mimic the structure of viruses. They are produced by expressing antigenic proteins that self-assemble into particles, which are then taken up by dendritic cells to activate the adaptive immune response. Their non-infectious nature, virus-like structure, and high immunogenicity make them a promising vaccine development platform.
VLPs have been utilized in several commercially available vaccines, such as those against hepatitis B virus (HBV) and human papillomavirus (HPV), and are notable for their ability to induce both innate and adaptive immune responses. The significance of VLPs lies in their potential to revolutionize vaccine development by offering a reliable and cost-effective platform that can be rapidly produced and tailored for various diseases.1,2
Streamlining Production Through Cell-Free Expression
The development of VLP-based vaccines is a complex process, fraught with technical challenges. These challenges can lead to issues with yield and assembly, hindering their wider adoption as a commercially viable vaccine platform.
- VLP expression: Optimal cell concentration, multiplicity of infection, and time of harvest are crucial for protein expression and VLP assembly.5
- VLP assembly: In vivo assembly of VLPs involves complex interactions between capsid proteins, requiring optimal conditions for successful assembly.5
- Upstream processing: Non-optimal process parameters like dissolved oxygen concentration, pH, temperature, etc., can hinder cell growth or interfere with post-translational modification mechanisms.5
- Downstream processing: Separating VLPs from a complex mixture of proteins can be challenging due to similar sizes and molecular weights, and protein aggregation can lead to loss of target protein or reduced immunogenicity.5
Given the numerous challenges in VLP-based vaccine development, selecting a suitable production platform that ensures stable expression and assembly and scale-up to larger production volumes is crucial. Flexible and scalable cell-free systems could offer a solution by significantly reducing the time-to-clinic, thereby enhancing the prospects of successful VLP-based vaccine production.
Here, we show the successful expression, assembly, and scaled-up production of immunogenic HBc VLPs in the eukaryotic cell-free expression system ALiCE®.
We also demonstrate the ability to conjugate antigens to preassembled VLPs for rapid adaptation to different pathogens.6,7
Materials and Methods
Optimizing VLP Expression with the ALiCE® Cell-Free Platform
Three variations of HBc VLP sequences were tested:
VLP-1: Untagged full-length HBc VLP sequence
VLP-2: Full-length HBc sequence with N-terminal Strep-II tag
VLP-3: Full-length HBc with Strep-II tag on spike sequence
The pALiCE constructs were prepared and expression reaction set up with the following parameters:
VLPs were isolated via affinity chromatography using Ni-NTA.
- As the HBc VLP monomers contain neither membrane spanning domains or ER-/Golgi-mediated post-translational modifications (PTMs), the sequences were cloned into the pALiCE01 vector 6
- DNA was added to the ALiCE® reaction mix to a final concentration of 5 nM.
- Initial 50 µL ALiCE® reactions were set up according to user manual guidelines, and the expression reaction run for 48 hours. The best-expressing construct was then selected for scaled protein expression in ALiCE lysate volumes ranging from 0.1 mL to 1000 mL.
KEY OPTIMZATION STRATEGY
Purification of VLPs presents some challenges. In the case of expression in ALiCE®, the lysate background characteristics (e.g. presence of nucleic acids) needs to be considered, together with the properties of your VLP (e.g. hydrophobicity, charge, and charge location within the VLP structure). We recommend a multimodal resin such as Capto Core as a first step, followed by a polishing step tailored to the specific properties of your VLP.
Expressing the Influenza RBD with ALiCE®
The receptor binding domain of the influenza virus was cloned and expressed in a separate ALiCE reaction:
- The influenza RBD protein was purified by affinity chromatography using the Strep-II-tag.
- The selected influenza RBD lacks membrane spanning domains and PTMs, so was also cloned into the pALiCE02 vector, together with a Strep II Tag.
- Expression was performed in a 2 mL reaction volume run for 48 hours.
RESULTS
Successful expression and assembly of VLPs6,7
Following microsomal disruption, we chose fractionation instead of the faster one-step protocol available in the ALiCE user manual. We recommend fractionation to obtain ultrapure proteins suitable for downstream applications, such as binding assays.
- ALiCE® enabled the expression of various VLP constructs within 48 hours.
- All three HBc VLP constructs produced high protein yields with expected sizes.
- The subsequent dot blot assay (and TEM analysis; data not shown) show proper assembly of two out of the three VLPs, highlighting the capability of ALiCE for rapid screening and characterization of VLPs.
Robust immune response by HBc VLPs6,7
- VLPs produced in ALiCE® were recognized and internalized by human myeloid immature dendritic cells, a crucial step in the initiation of an adaptive immune response.
- HBc VLPs also triggered the synthesis of pro-inflammatory cytokines (data not shown).
- The results show that VLPs produced in ALiCE® can trigger a strong immune response, overcoming a significant barrier to producing VLP-based vaccines in a cell-free system.
Simple scale-up of HBc VLPs
- Linear scale-up of an HBc VLP in a 20,000-fold range was successfully achieved using the ALiCE lysate.
- Structural integrity after scale-up was confirmed by TEM analysis (data not shown).
»A 1000 ml ALiCE reaction would produce a～0.4 g of a vaccine candidate. This would equal ～ 8,000 to 100,000 vaccine doses in just 48 hours, making ALiCE the first ever eukaryotic CFPS to be scaled in batch mode.«
Jorge Armero Giménez, Platform Development Scientist, LenioBi
Antigen conjugation of cell-free VLPs
- The receptor binding domain (RBD) of the influenza virus was successfully expressed and conjugated to HBc VLP.
- The TEM results show a homogenous coating of VLPs with target antigens.
- The ability to conjugate pathogen-specific antigens to pre-assembled carrier VLPs exemplifies the versatility and adaptability of the ALiCE platform for vaccine production.
KEY BENEFITS OF ALiCE®
VLPs are a promising alternative to traditional vaccine platforms as they trigger a strong and lasting immune response, with fewer safety concerns and higher stability than existing vaccine technologies. The ALiCE® cell-free expression platform, with its synthesis speed, antigen-conjugation capability, immunogenicity, and scalability, has immense potential as a versatile pandemic-preparedness vaccination platform.
Removing cell-line development as a bottleneck in pandemic response
The ALiCE® eukaryotic cell-free protein expression system is capable of producing even the most complex proteins in under 48 hours. This shaves off months of cell line development in VLP-based vaccine production, making it a promising platform to rapidly screen and validate VLPs of clinical relevance, thereby accelerating time to approval.
Versatility to produce antigen-conjugated, immunogenic vaccine candidates
Unlike traditional cell-based expression systems, which necessitate extensive screening of multiple constructs to identify those that correctly assemble into immunogenic VLPs, ALiCE® offers a more efficient solution. This system can be readily adapted to express and screen antigen-conjugated VLPs, significantly reducing the time required for these processes.
Moreover, the quality of the VLPs produced using ALiCE®, as shown by TEM analysis and immune response assays, demonstrates its effectiveness as a VLP factory. This makes ALiCE® a versatile and reliable platform for vaccine production in response to evolving threats.
Scalable development of vaccine candidates
The challenge of scaling eukaryotic cell-free systems has hindered their widespread adoption until now. ALiCE® operates efficiently from microliter reactions in microtiter plates to liter-scale reactions in bioreactors, demonstrating optimal performance at all scales. This scalability has been validated here with a VLP candidate. Currently, ALiCE® is being evaluated for the manufacturing of vaccine clinical trial material with support from CEPI.
References
Brisse M, Vrba SM, Kirk N, Liang Y, Ly H. Emerging Concepts and Technologies in Vaccine Development. Front Immunol. 2020;11:583077.
Mohsen MO, Zha L, Cabral-Miranda G, Bachmann MF. Major findings and recent advances in virus-like particle (VLP)-based vaccines. Semin Immunol. 2017;34:123-132.
Armero-Gimenez J, Wilbers R, Schots A, Williams C, Finnern R. Rapid screening and scaled manufacture of immunogenic virus-like particles in a tobacco BY-2 cell-free protein synthesis system. Front Immunol. 2023;14:1088852.
Peyret H, Gehin A, Thuenemann EC, et al. Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins. PLoS One. 2015;10(4):e0120751.
Gupta R, Arora K, Roy SS, et al. Platforms, advances, and technical challenges in virus-like particles-based vaccines. Front Immunol. 2023;14:1123805.
LenioBio Application Note: Expressing Virus-Like Particles using the ALiCE Cell-Free Protein Expression System
LenioBio Poster: Rapid screening and scaled manufacture of immunogenic virus-like particles in the ALiCE cell-free protein synthesis system
LenioBio Press Release: https://www.leniobio.com/press-release/plant-based-alice-technology-could-shave-weeks-off-vaccine-production/