Laura Cervera

HIV-1 Gag virus-like particles (VLPs) have been drawing attention as vaccine platform for their non-infectivity, ability to induce robust immune responses and versatility. However, challenges in their production, purification, and preservation still hinder their application. The production process, often reliant on transient gene expression (TGE), faces scalability limitations. Moreover, the downstream processing (DSP) presents challenges, including separating VLPs from extracellular vesicles (EVs), scale-up, and the lack of analytical methods to monitor the entire process. A complete bioprocess for Gag VLPs production is presented here, combining perfusion-based upstream production with a three-step DSP. Perfusion-based continuous production of Gag VLPs demonstrated significant yield improvements, with a 2.4-fold increase in VLP volumetric productivity compared to previous methods. Subsequent DSP steps, including secondary clarification and anion exchange chromatography (AEC) capture, resulted in approximately 60% recovery and purity. This ensures its scalability potential and robustness. The post-purification lyophilization step maintained VLP integrity and stability. This study presents an intensification strategy to address critical challenges in Gag VLP production, purification, and preservation, offering insights into enhancing vaccine biomanufacturing and distribution.

Virus-like particles (VLPs) are non-infective vaccine candidates that have gained interest given their natural ability to elicit strong immune responses. Particularly, HIV-1 Gag-based VLPs are one of the most described platforms for vaccine development, provided their ability for successful pseudotyping either by genetic engineering or click chemistry. When Gag polyprotein is recombinantly expressed, VLPs are naturally assembled in the vicinity of the cell membrane and then secreted by cell budding, taking part of the host cell membrane. Their properties are dependent upon the cell line and manufacturing method. Although great advancements toward the implementation of analytical methods have been made, VLP quality attributes are quite unclear whenever production is enhanced by metabolic engineering or process intensification strategies. This work offers a comparative study of VLP quality attributes upon transient gene expression (TGE) in HEK293 cell cultures operated in batch and perfusion mode. Moreover, the impact of specific productivity is also studied by ataxia telangiectasia mutated (ATM) gene silencing, which has been reported to enhance fourfold VLP production. A linear negative correlation was found between the ratio of Gag monomers/VLP and specific productivity. 3100 ± 100 monomers/VLP were obtained for the standard batch production, dropping to 1900 ± 100 and 800 ± 60 for the perfusion and batch ATM-knockdown conditions, respectively. Furthermore, functionalization rates were measured in terms of Cy5 per total particles (TP). Both perfusion-derived nanoparticles achieved functionalization rates of 2800 Cy5/TP. On the contrary, those nanoparticles produced in batch yielded functionalization rates below 1000 Cy5/TP. Moreover, a complete lipidome analysis revealed a relative decrease in the quantity of lipid/particle for all studied conditions in comparison to the standard batch production. Finally, all VLP samples were characterized to assess the impact of the differential physicochemical properties upon purification and stability rates. Key points: • VLP quality inversely correlates with Gag-specific productivity and operation mode. • Functionalization and lipid content drop with metabolic burden or ATM silencing. • Perfusion enables high VLP recovery and lyophilization with preserved morphology.

Enveloped nanoparticles such as extracellular vesicles (EVs) and virus-like particles (VLPs) have emerged as promising nanocarriers capable of transporting bioactive molecules for drug delivery and vaccination. Optimized functionalization methodologies are required to increase the functionalization levels of these nanoparticles, enhancing their performance. Here, a bioorthogonal copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) reaction has been optimized to functionalize human immunodeficiency virus type 1 (HIV-1) Gag-based VLPs and EVs. The optimization process has been carried out through reaction kinetics and design of experiments (DoE) using Cy5 as a reporter molecule. The functionalization of both VLPs and EVs has been studied using super-resolution fluorescence microscopy (SRFM), revealing remarkable differences between Gag-VLPs and coproduced EVs. EVs produced by mock transfection and cell growth have been functionalized achieving a mean of 3618.63 ± 48.91 and 6498.75 ± 352.71 Cy5 molecules covalently linked per particle (Cy5_cov/particle), respectively. Different nanoparticles have been functionalized with two linear B-cell epitopes from the Spike protein of SARS-CoV-2, S_315-338 TSNFRVQPTESIVRFPNITNLCPF and S_648-663 GCLIGAEHVNNSYECD, and analyzed by an immunoassay with sera from COVID-19 patients. The obtained results validate the selected B-cell epitopes and highlight the potential of the optimized functionalization approach for the development of nanoparticle-based vaccines.

Several strategies have been developed in recent years to improve virus-like particle (VLP)-based vaccine production processes. Among these, the metabolic engineering of cell lines has been one of the most promising approaches. Based on previous work and a proteomic analysis of HEK293 cells producing Human Immunodeficiency Virus-1 (HIV-1) Gag VLPs under transient transfection, four proteins susceptible of enhancing VLP production were identified: ataxia telangiectasia mutated (ATM), ataxia telangiectasia and rad3-related (ATR), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit delta (PDEδ). The knockdown of ATM, ATR, and PDEδ in HEK293 cells increased HIV-1 VLP titers in the supernatant by 3.4-, 2.1-, and 2.2-fold, respectively. Also, possible metabolic synergies between plasmids were investigated by statistical design of experiments (DoE), enabling us to identify the optimal production strategy, that was further demonstrated at lab-scale stirred tank bioreactor operated in perfusion, significantly increasing both VLPs specific and volumetric productivities to 8.3 × 10 ³ VLPs/cellxday and 7.5 × 10 ¹² VLPs/Lxday, respectively. Key points: • ATM, ATR, and PDEδ knockdowns increased VLP production in HEK293 cells. • Knockdown of ATM increased budding efficiency and extracellular vesicle concentration. • ATM knockdown could be intensified to bioreactor scale operated in perfusion. Graphical

The robustness, safety, versatility, and high immunogenicity of virus-like particles (VLPs) make them a promising approach for the generation of vaccines against a broad range of pathogens. VLPs are recombinant macromolecular structures that closely mimic the native conformation of viruses without carrying viral genetic material. Particularly, HIV-1 Gag-based VLPs are a suitable platform for the presentation of the SARS-CoV-2 Spike (S) protein on their surface. In this context, this work studies the effect of different rationally engineered mutations of the S protein to improve some of its characteristics. The studied variants harbored mutations such as proline substitutions for S stabilization, D614G from the early dominant pandemic form, the elimination of the S1/S2 furin cleavage site to improve S homogeneity, the suppression of a retention motif to favor its membrane localization, and cysteine substitutions to increase its immunogenicity and avoid potential undesired antibody-dependent enhancement (ADE) effects. The influence of the mutations on VLP expression was studied, as well as their immunogenic potential, by testing the recognition of the generated VLP variants by COVID-19 convalescent patients’ sera. The results of this work are conceived to give insights on the selection of S protein candidates for their use as immunogens and to showcase the potential of VLPs as carriers for antigen presentation.

This section introduces the content of the course, provides a brief introduction to the 12 chapters of this reference textbook, and gives a brief overview of the value of vaccines as a commodity essential for public health. It also provides an overview of the key stakeholders in the vaccine field and global market. The core content of the book is distributed in chapters that cover the i) the vaccine design supported by a summary of the key concepts in virology and immunology; ii) the upstream processing of viral vaccines, including process intensification concepts; iii) the downstream processing with specific applications of purification and polishing steps to viral structures; iv) the analyses of the critical quality attributes of the viral product as well as the process analytical technologies that support the product release and process robustness validation; v) a series of case studies presenting licensed inactivated or attenuated vaccines (influenza vaccines), advanced vaccine technology platforms (virus vectored and virus like particles vaccines) and an update on licensed vaccines for the COVID-19 pandemic, including mRNA vaccines.

Virus‐like particles (VLPs) resemble the viral native conformation by the recombinant expression of their structural proteins lacking the genetic material making them very safe. Their highly organized and repetitive structure has proven that they generate a potent immunogenic response activating both cellular and humoral immunogenicity responses. There is a large diversity of VLP configurations: from the simplest non‐enveloped single‐protein structure, such as in the Hepatitis B, to multilayered protein configurations and enveloped VLPs. Enveloped VLPs consist of one or more structural protein surrounded by the producer cell membrane that is acquired when the VLP buds from the cell. If there are antigens expressed at the producer cell membrane, when it buds, they are incorporated at the surface of the VLP. The ease of production, safety and their efficient recognition and cellular uptake, has expanded the interest on the possible applications of these structures in the last decade. VLPs can be further improved by encapsulation, chemical conjugation, and genetic manipulation. Bioengineering has been applied to strengthen their stability and immunostimulatory properties and to generate novel engineered VLPs, as well as vectors for DNA and drug delivery strategies. In this chapter an example of Gag based enveloped VLPs will be discussed.