Platform Process for an Autonomous Production of Virus-like Particles.

Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people, and smaller outbreaks have since occurred in Bangladesh and India. In this study, we have defined, for the first time, host factors that interact with henipavirus M proteins and contribute to viral particle assembly. We have also defined a new host protein-viral protein binding interface that can potentially be targeted for the inhibition of paramyxovirus infections.

This work aimed to assess the SARS-CoV-2 structural proteins' expression and virus-like particles (VLP) production by Baculovirus/Insect cell platform using two levels of Multiplicity of Infection (MOI), and two culture media, one of them a serum-free medium and the other one chemically defined. Two SARS-CoV-2 VLP were obtained from Sf9 cells coinfection using in both cases, three monocistronic recombinant baculoviruses holding the genes of Nucleocapsid (N; MOI = 2 or 0.2), Membrane (M; MOI = 1 or 0.1), and Envelope (E; MOI = 1 or 0.1) viral proteins, and the fourth one was changed between a baculovirus bearing Spike protein (S; MOI = 3 or 0.3) or receptor-binding domain (RBD; MOI = 3 or 0.3) genes of SARS-CoV-2. Similar performance was verified for both culture media in SARS-CoV-2 VLP production bearing four structural virus proteins or RBD domain. The SARS-CoV-2 structural proteins' expression was comparable at different MOIs (tenfold) as well as SARS-CoV-2 VLP size (around 100nm). The increase in specific death rates over the coinfection phase was confirmed in relatively high MOI assays. This finding was related to an exponential virus titer profile for high MOIs over the entire infection phase, meanwhile, a viral peak was observed at low MOIs, confirming a secondary infection. The SARS-CoV-2 VLP improved production carrying immunogenic S protein was confirmed concerning others holding RBD. However, the protein composition of produced VLP should be studied further to assess the VLP homogeneity when different culture media and MOIs are used.

Ebola virus (EBOV) VP40 is a major driving force of nascent virion production and a negative regulator of genome replication/transcription. Here, we showed that the YIGL sequence at the C-terminus of EBOV VP40 is important for virus-like particle (VLP) production and the regulation of genome replication/transcription. Accordingly, a mutation in the YIGL sequence caused defects in VLP production and genome replication/transcription. The residues I293 and L295 in the YIGL sequence were particularly critical for VLP production. Furthermore, an in silico analysis indicated that the amino acids surrounding the YIGL sequence contribute to intramolecular interactions within VP40. Among those surrounding residues, F209 was shown to be critical for VLP production. These results suggested that the VP40 YIGL sequence regulates two different viral replication steps, VLP production and genome replication/transcription, and the nearby residue F209 influences VLP production.

Virus-like particles (VLPs), nanoscale self-assembled structures lacking viral genetic material, have emerged as a versatile platform for vaccines, targeted delivery systems, and gene-editing applications owing to their strong immunogenicity, favorable biosafety profile, and high engineerability. However, the complex architecture of VLPs, their significant size heterogeneity, and the diversity of process- and product-related impurities generated in different expression systems make downstream purification a major bottleneck limiting product quality, yield, and manufacturability. This review systematically discusses advanced downstream purification strategies for VLPs from the perspective of three major objectives: preservation of structure and biological activity, control of product heterogeneity, and assurance of viral safety. First, strategies for maintaining VLP integrity and function are examined, including optimization of solution conditions, adoption of gentle yet efficient separation operations, and integration of process analytical technology (PAT) to reduce process-induced damage. Second, the review summarizes multi-step purification approaches-spanning clarification, ultrafiltration/diafiltration (UF/DF), chromatography, and disassembly/reassembly-to remove host cell proteins, host cell DNA, and product-related impurities while improving particle homogeneity and stability. Third, viral safety is discussed primarily from the perspective of downstream virus clearance under host-dependent risk, with particular attention to orthogonal clearance steps tailored to VLP properties and expression systems such as CHO cells and insect cell-baculovirus platforms. Overall, this review provides a CQA-oriented framework and practical guidance for the development of robust, scalable, and GMP-compliant downstream purification processes for VLP-based products.

Rotavirus is the major enteric pathogen that is responsible for more than 500,000 deaths of children worldwide annually. The use of live-attenuated oral rotavirus vaccines has significantly reduced child mortality rate in developed countries. However, the situation still remains unacceptably worse in most developing countries due to low efficacy of the vaccines in the region. The vaccines often do not reach the developing countries due to financial and logistic challenges. A high burden of rotavirus disease and the unresolved challenges with the current rotavirus vaccines, particularly in developing countries, have ignited efforts to develop next-generation low-cost, effective and safe vaccine candidates. Virus-like particles (VLPs) have come into focus for their promising application in vaccination because of their unique and attractive set of properties including safety, self-adjuvanticity, developability, economy and the ability to be formulated for stability without a cold chain. Their impact on human health is already evident through commercialization of VLP vaccines against hepatitis B virus infection, human papillomavirus-induced cervical cancer and hepatitis E virus infection. Rotavirus-like particles (RLPs) with proven preclinical immunogenicity and protective efficacy are considered as safe and effective vaccine candidates. While rotavirus-like particles comprising multiple viral proteins can be difficult to process, modular VLPs presenting rotavirus antigenic modules are promising approaches in reducing process complexity and cost. Depending on the physicochemical properties, size and /or surface density of modules, modularization may affect production of stable VLP forming subunits, termed capsomeres, and prevent VLP assembly. This thesis demonstrates a multipronged approach for a low-cost production of stable bacterially-produced modular murine polyomavirus capsomeres and in vitro assembled VLPs presenting a rotavirus highly conserved peptide epitope (RV10) and a conformational and a virus neutralizing 18 kDa antigen (VP8*), separately. The experimental works in this thesis were carried out to address challenges associated with the adverse effects of inserted module’s physicochemical properties, size and density on the production of modular capsomeres and VLPs. Particularly, the expression and purification of stable modular capsomeres for in vitro modular VLP assembly and the enhancement of module-specific immune response were investigated. The outcomes of the work in this thesis are: (i) a rapid and simple high-throughput screening method based on dynamic light scattering measurements was developed to identify additives for enhanced stability of modular capsomeres; (ii) using synthetic biology designs, the hydrophobicity of RV10 modules was engineered for enhanced stability of modular capsomeres; (iii) in vitro assembled modular VLPs displaying RV10 modules were obtained via a module titration approach using Escherichia coli protein co-expression strategy; and (iv) highly stable and immunogenic modular VLPs displaying conformational VP8* were produced using a combination of Escherichia coli protein co-expression, simplified modular capsomere purification steps and cell-free in vitro VLP assembly. The outcomes of this thesis expand the potential and plasticity of the murine polyomavirus VLP platform for presentation of antigenic modules regardless of their adverse physicochemical properties and large size. This work introduces, to the best of our knowledge, the first bacterially-produced capsomeres and in vitro assembled VLPs displaying either hydrophobic RV10 peptide modules or 18 kDa rotavirus VP8* large antigens. The modular capsomeres (CapVP8*) and modular VLPs (VLP-VP8*) induced high levels of VP8*-specific antibodies in mice. The high immunogenicity of CapVP8* and VLP-VP8*likely indicates protective efficacy and makes them a more viable vaccine candidate for further development to prevent rotavirus in the developing world at affordable cost. The strategies developed in this thesis potentially provides a cost-effective production route for modular capsomere and VLPs presenting other antigenic modules from rotavirus and other target pathogens, highly suitable to manufacturing economics for the developing world.

hRSV is responsible for >100,000 deaths in children worldwide, and a vaccine is not available. Among the potential anti-hRSV approaches are virus-like particle (VLP) vaccines, which, based on resemblance to virus or viral components, can induce protective immunity. For hRSV, few reports are available concerning authentic VLP production or testing, in large part because VLP production is inefficient and the mechanisms underlying particle assembly are poorly understood. Here, we took advantage of the cell-associated nature of RSV particles and used high-resolution microscopy analyses to examine the viral proteins required for formation of wild-type-virus-resembling VLPs, the contributions of these proteins to morphology, and the domains involved in incorporation of the antigenically important viral F protein. The results provide new insights that will facilitate future production of hRSV VLPs with defined shapes and compositions and may translate into improved manufacture of live-attenuated hRSV vaccines.

Biopharma Is Going Digital … Bit by Bit

Lassa virus (LASV) small zinc-finger protein (Z), which contains two L-domain motifs, plays a central role in virus budding. Here, we report that co-expression of glycoprotein (GPC) altered the requirements for cholesterol but not the L-domains and host factor, Tsg101, for Z-induced virus-like particle (VLP) production. In particular, the cholesterol requirement for VLP production was cell-type-dependent. In addition, GPC was found to be important for co-localization of Z with CD63, a late endosomal marker. We also found that the N-terminal region (aa 3-10) of Z was critical for its myristoylation and VLP production. These findings will contribute to our understanding of LASV assembly and budding.

SARS-CoV-2 is an RNA enveloped virus responsible for the COVID-19 pandemic that conducted in 6 million deaths worldwide so far. SARS-CoV-2 particles are mainly composed of the 4 main structural proteins M, N, E and S to form 100 nm diameter viral particles. Based on productive assays, we propose an optimal transfected plasmid ratio mimicking the viral RNA ratio in infected cells. This allows SARS-CoV-2 Virus-Like Particle (VLPs) formation composed of the viral structural proteins M, N, E and mature S. Furthermore, fluorescent or photoconvertible VLPs were generated by adding a fluorescent protein tag on N or M mixing with unlabeled viral proteins and characterized by western blots, atomic force microscopy coupled to fluorescence and immuno-spotting. Thanks to live fluorescence and super-resolution microscopies, we quantified VLPs size and concentration. SARS-CoV-2 VLPs present a diameter of 110 and 140 nm respectively for MNE-VLPs and MNES-VLPs with a concentration of 10e12 VLP/ml. In this condition, we were able to establish the incorporation of the Spike in the fluorescent VLPs. Finally, the Spike functionality was assessed by monitoring fluorescent MNES-VLPs docking and internalization in human pulmonary cells expressing or not the receptor hACE2. Results show a preferential maturation of S on N(GFP) labeled VLPs and an hACE2-dependent VLP internalization and a potential fusion in host cells. This work provides new insights on the use of non-fluorescent and fluorescent VLPs to study and visualize the SARS-CoV-2 viral life cycle in a safe environment (BSL-2 instead of BSL-3). Moreover, optimized SARS-CoV-2 VLP production can be further adapted to vaccine design strategies.

Virus-like particles (VLPs) are spontaneously generated from viral capsid proteins. VLPs imitate genuine viruses visually and physiologically, but lack viral DNA. Various VLP designs provide structural and functional appeal. Spontaneous polymerization of viral capsid proteins may result in the formation of VLPs with geometrical symmetry, which are often icosahedral, spherical, or rod-like. Moreover, functionalized VLPs may precisely target cancer cells and recruit macrophages to destroy them. The ability to target tumors for therapeutic drug delivery through VLP-based delivery platforms in novel and intriguing aspects related to cancer treatment is the primary goal of VLP design. Cancer therapies require precise targeting of diagnostic or therapeutic elements to tumor cells while avoiding healthy cells and tissues. VLPs offer an innovative approach as site-specific drug delivery systems, reducing systemic toxicity and minimizing injury to healthy cells. Immunotherapy, which boosts the host's immune system, has fewer side effects. Cancer vaccines aim to induce an immune response that provides protection against tumor cells. Due to their naturally fitted particle size and repetitive structural order, VLPs may be employed as a vaccine without any adjuvant. Recombinant VLP structures can be enhanced by including antigenic epitopes of viruses or different disease-related antigens, and targeting peptides to the interior and exterior surfaces, making them potential tools for future immunizations with preventive and regenerative qualities. Additionally, VLP-based delivery strategies may enhance immunogenicity and provide a more effective and safer approach to managing solid cancers with fewer side effects compared to chemotherapy or radiation. However, the production of chimeric VLPs still faces challenges, such as the need for more reliable preclinical animal models and associated costs. Despite these obstacles, ongoing research will improve VLP-based technologies and increase their potential advantages. This review aims to provide basic information on VLPs and outline current studies on their use as drug and vaccine delivery systems in different cancers, highlighting their potential as a promising cancer treatment strategy. The key terms in the literature search-including drug delivery, gene therapy, multi-capsid VLPs, and virus-like particles (VLPs)-were searched in international databases, namely Web of Science, PubMed, and Scopus from 2003 to 2022.

COVID-19 is an infectious disease resulting from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged as a global pandemic in 2020. Virus-like particles (VLP) are artificially generated nanoparticles composed of a subset of viral components that closely mimic the native virus in size, structure, and surface composition. These features contribute to their robust immunogenic potential, stimulating both antibody-mediated and cell-mediated immune responses. This study aimed to evaluate the expression of SARS-CoV-2 structural proteins and the VLP production using a baculovirus–insect cell system. Two monocistronic recombinant baculoviruses, each carrying either the spike (S) or nucleocapsid (N) gene of SARS-CoV-2, were employed in two different culture systems: Schott flasks and a stirred-tank bioreactor. The Sf9 cells showed substantial differences between the Schott flask and stirred tank bioreactor in terms of metabolism, virus titer, and cell death after infection. Both proteins’ expressions were confirmed in the two culture systems assessed. However, VLP formation (69.1–78.2 nm) was only confirmed in the co-infection setup with both monocistronic recombinant baculoviruses. In contrast, infection with only the S-protein baculovirus failed to produce VLP in our findings.

As demonstrated by the 2015 Zika virus outbreak in the Americas, emerging and re-emerging arboviruses are public health threats that warrant research investment for the development of effective prophylactics and therapeutics. Many arboviral diseases are underreported, neglected, or of low prevalence, yet they all have the potential to cause outbreaks of local and international concern. Here, we show the production of virus-like particles (VLPs) using a rapid and efficient recombinant vaccinia virus (VACV) expression system for five tick- and mosquito-borne arboviruses: Powassan virus (POWV), Heartland virus (HRTV), severe fever with thrombocytopenia syndrome virus (SFTSV), Bourbon virus (BRBV) and Mayaro virus (MAYV). We detected the expression of arbovirus genes of interest by Western blot and observed the expression of VLPs that resemble native virions under transmission electron microscopy. We were also able to improve the secretion of POWV VLPs by modifying the signal sequence within the capsid gene. This study describes the use of a rapid VACV platform for the production and purification of arbovirus VLPs that can be used as subunit or vectored vaccines, and provides insights into the selection of arbovirus genes for VLP formation and genetic modifications to improve VLP secretion and yield.

The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus-like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti-myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high-performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038-1045, 2016.

Infectious bronchitis (IB) is a highly contagious, acute respiratory disease in chickens, with a severe economic impact on poultry production globally. The rapid emergence of regional variants of this Gammacoronavirus warrants new vaccine approaches that are more humane and rapid to produce than the current embryonated chicken egg-based method used for IB variant vaccine propagation (chemically-inactivated whole viruses). The production of virus-like particles (VLPs) expressing the Spike (S) glycoprotein, the major antigen which induces neutralizing antibodies, has not been achieved in planta up until now. In this study, using the Agrobacterium-mediated Nicotiana benthamiana (tobacco plant) transient expression system, the highest levels of VLPs displaying a modified S protein of a QX-like IB variant were obtained when the native transmembrane (TM) domain and cytoplasmic tail were substituted with that of the Newcastle disease virus (NDV) fusion glycoprotein, co-infiltrated with the NDV Matrix protein. In comparison, the native IB modified S co-infiltrated with IB virus membrane, envelope and nucleocapsid proteins, or substituted with the TM and CT of an H6-subtype influenza A virus hemagglutinin glycoprotein yielded lower VLP expression levels. Strong immunogenicity was confirmed in specific pathogen free chickens immunized intramuscularly with VLPs adjuvanted with Emulsigen®-P, where birds that received doses of 5 μg or 20 μg (S protein content) seroconverted after two weeks with mean hemaggluttination inhibition titres of 9.1 and 10 log2, respectively. Plant-produced IB VLP variant vaccines are safer, more rapid and cost effective to produce than VLPs produced in insect cell expression systems or the traditional egg-produced inactivated whole virus oil emulsion vaccines currently in use, with great potential for improved IB disease control in future.

Supplying SARS-COVID-19 vaccines in quantities to meet global demand has a bottleneck in manufacturing capacity. Assessment of existing mRNA (messenger ribonucleic acid) vaccine processing shows the need for digital twins enabled by process analytical technology approaches to improve process transfers for manufacturing capacity multiplication, reduction of out-of-specification batch failures, qualified personnel training for faster validation and efficient operation, optimal utilization of scarce buffers and chemicals, and faster product release. A digital twin of the total pDNA (plasmid deoxyribonucleic acid) to mRNA process is proposed. In addition, a first feasibility of multisensory process analytical technology (PAT) is shown. Process performance characteristics are derived as results and evaluated regarding manufacturing technology bottlenecks. Potential improvements could be pointed out such as dilution reduction in lysis, and potential reduction of necessary chromatography steps. 1 g pDNA may lead to about 30 g mRNA. This shifts the bottleneck towards the mRNA processing step, which points out co-transcriptional capping as a preferred option to reduce the number of purification steps. Purity demands are fulfilled by a combination of mixed-mode and reversed-phase chromatography as established unit operations on a higher industrial readiness level than e.g., precipitation and ethanol-chloroform extraction. As a final step, lyophilization was chosen for stability, storage and transportation logistics. Alternative process units like UF/DF (ultra-/diafiltration) integration would allow the adjustment of final concentration and buffer composition before lipid-nano particle (LNP) formulation. The complete digital twin is proposed for further validation in manufacturing scale and utilization in process optimization and manufacturing operations. The first PAT results should be followed by detailed investigation of different batches and processing steps in order to implement this strategy for process control and reliable, efficient operation.