Production Of Virus-like Particles Research Articles

A Replicating Viral Vector Greatly Enhances Accumulation of Helical Virus-Like Particles in Plants.

The production of plant helical virus-like particles (VLPs) via plant-based expression has been problematic with previous studies suggesting that an RNA scaffold may be necessary for their efficient production. To examine this, we compared the accumulation of VLPs from two potexviruses, papaya mosaic virus and alternanthera mosaic virus (AltMV), when the coat proteins were expressed from a replicating potato virus X- based vector (pEff) and a non-replicating vector (pEAQ-HT). Significantly greater quantities of VLPs could be purified when pEff was used. The pEff system was also very efficient at producing VLPs of helical viruses from different virus families. Examination of the RNA content of AltMV and tobacco mosaic virus VLPs produced from pEff revealed the presence of vector-derived RNA sequences, suggesting that the replicating RNA acts as a scaffold for VLP assembly. Cryo-EM analysis of the AltMV VLPs showed they had a structure very similar to that of authentic potexvirus particles. Thus, we conclude that vectors generating replicating forms of RNA, such as pEff, are very efficient for producing helical VLPs.

SARS‐CoV‐2 Viral Budding and Entry can be Modeled Using BSL‐2 Level Virus‐Like Particles

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) was first discovered in December 2019 in Wuhan, China and expeditiously spread across the globe causing a global pandemic. Research on SARS‐CoV‐2, as well as the closely related SARS‐CoV‐1 and MERS coronaviruses is restricted to BSL‐3 facilities. Such BSL‐3 classification makes SARS‐CoV‐2 research inaccessible to the majority of functioning research laboratories in the US; this becomes problematic when the collective scientific effort needs to be focused on such in the face of a pandemic. However, a minimal system capable of recapitulating different steps of the viral life cycle without using the virus' genetic material could increase accessibility. In this work, we assessed the four structural proteins from SARS‐CoV‐2 for their ability to form virus‐like particles (VLPs) from human cells to form a competent system for BSL‐2 studies of SARS‐CoV‐2. After establishing the minimal system requirements for VLP production, we examined their morphological relevance with transmission electron microscopy (TEM). VLPs produced with all four viral structural proteins were approximately 100 nm in diameter and bared the characteristic coronavirus crown or ‘corona’. We next sought to evaluate the entry competency of our VLPs. GFP‐tagged VLPs were generated by fluorescently tagging one of the four structural proteins used to produce VLPs. Once incorporation of the GFP‐tagged protein into VLPs was confirmed, we used these GFP‐VLPs to infect HEK293 cells. GFP‐VLPs indeed did enter HEK293 cells and properly colocalized with endocytic markers Rab5 and LAMP1 in accordance with live virus data. To further evaluate viral entry, we made the VLP entry assay accessible to TEM analysis by replacing the GFP tag with an ascorbate peroxidase (APEX2) tag, which when oxidized produces a dark brown precipitate visible on micrographs. APEX2‐VLPs were found to be entry‐competent as well. In addition to entry, APEX2‐VLPs yield the ability to visualize VLP assembly at the ER‐Golgi intermediary complex (ERGIC) and for the first time we show localization of the structural proteins during SARS‐CoV‐2 VLP assembly, budding, and egress. In total, this research provides ample resources for other BSL‐2 laboratories interested in joining the growing field to try and understand SARS‐CoV‐2 assembly, budding, and entry dynamics, biochemical and biophysical questions on the four structural proteins, and drug screening of viral assembly, budding, and/or entry inhibitors.

Characterization of HIV-1 virus-like particles and determination of Gag stoichiometry for different production platforms.

The importance of developing new vaccine technologies towards versatile platforms that can cope with global virus outbreaks has been evidenced with the most recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Virus-like particles (VLPs) are a highly immunogenic, safe, and robust approach that can be used to base several vaccine candidates on. Particularly, HIV-1 Gag VLPs is a flexible system comprising a Gag core surrounded by a lipid bilayer that can be modified to present diverse types of membrane proteins or antigens against several diseases, like influenza, dengue, West Nile virus, or human papillomavirus, where it has been proven successful. The size distribution and structural characteristics of produced VLPs vary depending on the cell line used to produce them. In this study, we established an analytical method of characterization for the Gag protein core and clarified the current variability of Gag stoichiometry in HIV-1 VLPs depending on the cell-based production platform, directly determining the number of Gag molecules per VLP in each case. Three Gag peptides have been validated to quantify the number of monomers using parallel reaction monitoring, an accurate and fast, mass-spectrometry-based method that can be used to assess the quality of the produced Gag VLPs regardless of the cell line used. An average of 3617 ± 17 monomers per VLP was obtained for HEK293, substantially varying between platforms, including mammalian and insect cells. This offers a key advantage in quantification and quality control methods to characterize VLP production at a large scale to accelerate new recombinant vaccine production technologies.

In Vitro Assembly of Virus-Like Particles and Their Applications.

Virus-like particles (VLPs) are increasingly used for vaccine development and drug delivery. Assembly of VLPs from purified monomers in a chemically defined reaction is advantageous compared to in vivo assembly, because it avoids encapsidation of host-derived components and enables loading with added cargoes. This review provides an overview of ex cella VLP production methods focusing on capsid protein production, factors that impact the in vitro assembly, and approaches to characterize in vitro VLPs. The uses of in vitro produced VLPs as vaccines and for therapeutic delivery are also reported.

Assembly and Entry of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2): Evaluation Using Virus-Like Particles.

Research on infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is currently restricted to BSL-3 laboratories. SARS-CoV2 virus-like particles (VLPs) offer a BSL-1, replication-incompetent system that can be used to evaluate virus assembly and virus-cell entry processes in tractable cell culture conditions. Here, we describe a SARS-CoV2 VLP system that utilizes nanoluciferase (Nluc) fragment complementation to track assembly and entry. We utilized the system in two ways. Firstly, we investigated the requirements for VLP assembly. VLPs were produced by concomitant synthesis of three viral membrane proteins, spike (S), envelope (E), and matrix (M), along with the cytoplasmic nucleocapsid (N). We discovered that VLP production and secretion were highly dependent on N proteins. N proteins from related betacoronaviruses variably substituted for the homologous SARS-CoV2 N, and chimeric betacoronavirus N proteins effectively supported VLP production if they contained SARS-CoV2 N carboxy-terminal domains (CTD). This established the CTDs as critical features of virus particle assembly. Secondly, we utilized the system by investigating virus-cell entry. VLPs were produced with Nluc peptide fragments appended to E, M, or N proteins, with each subsequently inoculated into target cells expressing complementary Nluc fragments. Complementation into functional Nluc was used to assess virus-cell entry. We discovered that each of the VLPs were effective at monitoring virus-cell entry, to various extents, in ways that depended on host cell susceptibility factors. Overall, we have developed and utilized a VLP system that has proven useful in identifying SARS-CoV2 assembly and entry features.

Integrating high cell density cultures with adapted laboratory evolution for improved Gag‐HA virus‐like particles production in stable insect cell lines

Stable insect cell lines are emerging as an alternative to the insect cell-baculovirus expression vector system (IC-BEVS) for protein expression, benefiting from being a virus-free, nonlytic system. Still, the titers achieved are considerably lower. In this study, stable insect (Sf-9 and High Five) cells producing Gag virus-like particles (VLPs) were first adapted to grow under hypothermic culture conditions (22°C instead of standard 27°C), and then pseudotyped with a model membrane protein (influenza hemagglutinin [HA]) for expression of Gag-HA VLPs. Adaptation to lower temperature led to an increase in protein titers of up to 12-fold for p24 (as proxy for Gag-VLP) and sixfold for HA, with adapted Sf-9 cells outperforming High Five cells. Resulting Gag-HA VLPs producer Sf-9 cells were cultured to high cell densities, that is, 100 × 106 cell/ml, using perfusion (ATF® 2) in 1 L stirred-tank bioreactors. Specific p24 and HA production rates were similar to those of batch culture, enabling to increase volumetric titers by 7-8-fold without compromising the assembly of Gag-HA VLPs. Importantly, the antigen (HA) quantity in VLPs generated using stable adapted cells in perfusion was ≈5-fold higher than that from IC-BEVS, with the added benefit of being a baculovirus-free system. This study demonstrates the potential of combining stable expression in insect cells adapted to hypothermic culture conditions with perfusion for improving Gag-HA VLPs production.

Single dose of a replication-defective vaccinia virus expressing Zika virus-like particles is protective in mice

Zika virus (ZIKV), a flavivirus transmitted primarily by infected mosquitos, can cause neurological symptoms such as Guillian–Barré syndrome and microcephaly. We developed several vaccinia virus (VACV) vaccine candidates for ZIKV based on replication-inducible VACVs (vINDs) expressing ZIKV pre-membrane (prM) and envelope (E) proteins (vIND-ZIKVs). These vIND-ZIKVs contain elements of the tetracycline operon and replicate only in the presence of tetracyclines. The pool of vaccine candidates was narrowed to one vIND-ZIKV containing a novel mutation in the signal peptide of prM that led to higher expression and secretion of E and production of virus-like particles, which was then tested for safety, immunogenicity, and efficacy in mice. vIND-ZIKV grows to high titers in vitro in the presence of doxycycline (DOX) but is replication-defective in vivo in the absence of DOX, causing no weight loss in mice. C57BL/6 mice vaccinated once with vIND-ZIKV in the absence of DOX (as a replication-defective virus) developed robust levels of E-peptide-specific IFN-γ-secreting splenocytes and anti-E IgG titers, with modest levels of serum-neutralizing antibodies. Vaccinated mice treated with anti-IFNAR1 antibody were completely protected from ZIKV viremia post-challenge after a single dose of vIND-ZIKV. Furthermore, mice with prior immunity to VACV developed moderate anti-E IgG titers that increased after booster vaccination, and were protected from viremia only after two vaccinations with vIND-ZIKV.

Virus-Like Particles Produced Using the Brome Mosaic Virus Recombinant Capsid Protein Expressed in a Bacterial System.

Virus-like particles (VLPs), due to their nanoscale dimensions, presence of interior cavities, self-organization abilities and responsiveness to environmental changes, are of interest in the field of nanotechnology. Nevertheless, comprehensive knowledge of VLP self-assembly principles is incomplete. VLP formation is governed by two types of interactions: protein–cargo and protein–protein. These interactions can be modulated by the physicochemical properties of the surroundings. Here, we used brome mosaic virus (BMV) capsid protein produced in an E. coli expression system to study the impact of ionic strength, pH and encapsulated cargo on the assembly of VLPs and their features. We showed that empty VLP assembly strongly depends on pH whereas ionic strength of the buffer plays secondary but significant role. Comparison of VLPs containing tRNA and polystyrene sulfonic acid (PSS) revealed that the structured tRNA profoundly increases VLPs stability. We also designed and produced mutated BMV capsid proteins that formed VLPs showing altered diameters and stability compared to VLPs composed of unmodified proteins. We also observed that VLPs containing unstructured polyelectrolyte (PSS) adopt compact but not necessarily more stable structures. Thus, our methodology of VLP production allows for obtaining different VLP variants and their adjustment to the incorporated cargo.

Novel Virus-Like Particle Vaccine Encoding the Circumsporozoite Protein of Plasmodium falciparum Is Immunogenic and Induces Functional Antibody Responses in Mice.

RTS,S is the leading malaria vaccine in development, but has demonstrated only moderate protective efficacy in clinical trials. RTS,S is a virus-like particle (VLP) that uses the human hepatitis B virus as scaffold to display the malaria sporozoite antigen, circumsporozoite protein (CSP). Particle formation requires four-fold excess scaffold antigen, and as a result, CSP represents only a small portion of the final vaccine construct. Alternative VLP or nanoparticle platforms that reduce the amount of scaffold antigen and increase the amount of the target CSP antigen present in particles may enhance vaccine immunogenicity and efficacy. Here, we describe the production and characterization of a novel VLP that uses the small surface antigen (dS) of duck hepatitis B virus to display CSP. The CSP-dS fusion protein successfully formed VLPs without the need for excess scaffold antigen, and thus CSP represented a larger portion of the vaccine construct. CSP-dS formed large particles approximately 31-74 nm in size and were confirmed to display CSP on the surface. CSP-dS VLPs were highly immunogenic in mice and induced antibodies to multiple regions of CSP, even when administered at a lower vaccine dosage. Vaccine-induced antibodies demonstrated relevant functional activities, including Fc-dependent interactions with complement and Fcγ-receptors, previously identified as important in malaria immunity. Further, vaccine-induced antibodies had similar properties (epitope-specificity and avidity) to monoclonal antibodies that are protective in mouse models. Our novel platform to produce VLPs without excess scaffold protein has wide implications for the future development of vaccines for malaria and other infectious diseases.

Synthesis and characterization of human rotavirus A (Reoviridae: Sedoreovirinae: Rotavirus: Rotavirus A) virus-like particles

Rotavirus infection is the leading cause of acute gastroenteritis among infants. The development of new vaccines against rotavirus A is urgent because the virus has many genotypes, some of which have regional prevalence. Virus-like particles (VLP) is a promising way to create effective and safe vaccine preparations.The purpose of the study is to develop the technology for the production of VLP, containing VP2, VP4, VP6 and VP7 of viral genotypes prevalent on the territory of the Russian Federation, and to give its molecular genetic and virological characteristics. The virulent strain Wa G1P[8] of human RV A adapted to MARC-145 cell culture has been used. It was cultured and purified according to the method described by the authors earlier. Standard molecular genetic and cytological methods were used: gene synthesis; cloning into transfer plasmids; recombinant baculoviruses production in Bac-to-Bac expression system; VLP production in the insect cells; centrifugation in sucrose solution; enzyme-linked immunosorbent assay (ELISA); electron microscopy (EM); polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. VP4 and VP7 of the six most represented in Russia genotypes: G1, G2, G4, G9, P4, P8, as well as VP2 and VP6 were selected for VLP production. Recombinant baculoviruses were obtained with codon frequencies optimized for insect cells. Cabbage loopper (Trichoplusia ni) cell culture was coinfected with different combinations of baculoviruses, and VLP consisting of 2-4 proteins were produced. VLP were purified by centrifugation. The size and morphology of the particles matched the rotavirus A virion (by EM). The presence of rotavirus A proteins in VLP was confirmed by the ELISA, SDS-PAGE and western blot analysis. The technology for the synthesis of three-layer VLP consisting of VP2, VP4, VP6 and VP7 has been developed and optimized. The resulting VLP composition represents 6 serotypes of VP4 and VP7, which are most represented on the territory of Russia, and can be used for vaccine development.

Construction of a recombinant avipoxvirus expressing the env gene of Zika virus as a novel putative preventive vaccine

BackgroundZika virus (ZIKV) has been declared a public health emergency that requires development of an effective vaccine, as it might represent an international threat.MethodsHere, two novel DNA-based (pVAXzenv) and fowlpox-based (FPzenv) recombinant putative vaccine candidates were constructed that contained the cPrME genes of ZIKV. The env gene inserted into the fowlpox vector was verified for correct transgene expression by Western blotting and by immunofluorescence in different cell lines. The production of virus-like particles as a result of env gene expression was also demonstrated by electron microscopy. BALB/c mice were immunosuppressed with dexamethasone and immunized following a prime–boost strategy in a heterologous protocol where pVAXzenv was followed by FPzenv, to evaluate the immunogenicity of the Env protein. The mice underwent a challenge with an epidemic ZIKV after the last boost.ResultsThese data show that the ZIKV Env protein was correctly expressed in both normal human lung fibroblasts (MRC-5 cells) and green monkey kidney (Vero) cells infected with FPzenv, and that the transgene expression lasted for more than 2 weeks. After mucosal administration of FPzenv, the immunized mice showed specific and significantly higher humoral responses compared to the control mice. However, virus neutralizing antibodies were not detected using plaque reduction assays.ConclusionsAlthough BALB/c mice appear to be an adequate model for ZIKV infection, as it mimics the natural mild infection in human beings, inadequate immune suppression seemed to occur by dexamethasone and different immune suppression strategies should be applied before challenge to reveal any protection of the mice.

A simple method to purify recombinant HCV core protein expressed in Pichia pastoris for obtaining virus-like particles and producing monoclonal antibodies

In this study, we describe an optimized method of obtaining virus-like particles (VLPs) of the recombinant hepatitis C virus (HCV) core protein (HCcAg) expressed in yeast cells (Pichia pastoris), which can be used for the construction of diagnostic test systems and vaccine engineering. The described simplified procedure was developed to enable in vitro self-assembly of HCcAg molecules into VLPs during protein purification. In brief, the HCcAg protein was precipitated from yeast cell lysates with ammonium sulfate and renatured by gel filtration on Sephadex G-25 under reducing conditions. VLPs were self-assembled after the removal of the reducing agent by gel filtration on Sephadex G-25. Protein purity and specificity were evaluated by SDS-PAGE and immunoblotting analysis. The molecular mass of VLPs and their relative quantity were measured by HPLC, followed by confirmation of VLPs production and estimation of their shape and size by transmission electron microscopy. As a result, we obtained recombinant HCcAg preparation (with ~90% purity) in the form of VLPs and monomers, which has been used to produce hybridomas secreting monoclonal antibodies (mAbs) against HCcAg.

Metabolic engineering of HEK293 cells to improve transient transfection and cell budding of HIV-1 virus-like particles.

HIV-1 Gag virus-like particles (VLPs) are promising candidates for the development of future vaccines. Recent viral outbreaks have manifested the need of robust vaccine production platforms able to adapt to new challenges while achieving mass production capacity. For the rapid production of VLPs, the method of transient gene expression (TGE) have proved highly efficient. Based on a previous characterization of the HEK293 cell line upon transient transfection using multiplexed quantitative proteomics, molecular production bottlenecks and metabolic pathways likely to be optimized were identified. In this study, these molecular components and metabolic pathways have been explored and modulated via transient metabolic engineering using approaches like design of experiments to fully exploit and optimize VLP production, transfection and budding efficiency. Upon overexpression of endosomal sorting complex required for transport accessory proteins like NEDD4L and CIT, VLP production increased 3.3 and 2.9-fold, respectively. Overexpression of glycosphingolipid precursor enzyme UGCG improved transfection efficiency by 17% and knocking-down the Gag-binding protein CNP improved 2.5-fold VLP specific productivity. Combining CNP inhibition and UGCG overexpression further improved budding efficiency by 37.3%. Modulating VLP production and accessory pathways like intracellular budding, demonstrated the potential of metabolic engineering to optimize and intensify the development of robust production platforms for future vaccines.

Investigation of Kluyveromyces marxianus as a novel host for large\u2010scale production of porcine parvovirus virus\u2010like particles

BackgroundPorcine Parvovirus (PPV) is a Parvovirinae virus that can cause embryonic and fetal loss and death and mummification in affected fetal pigs. Unlike conventional vaccines, virus-like particles (VLPs) inherit the natural structure of their authentic virions and highly immunostimulatory that can induce strong humoral immune and T cell responses with no risk of pathogenicity. The production of PPV VLPs is still a challenge based on traditional expression platforms due to their low yields and high culture costs. Kluyveromyces marxianus is a safe and fast-growing eukaryote that can get high biomass with low-cost cultures. In this study, we investigated the expression and downstream processes of PPV VLPs in K. marxianus, and the potential for effective stand-alone vaccines.ResultsAfter optimization according to the codon bias of K. marxianus, the VP2 protein from Kresse strain was highly expressed. In a 5 L fermentator, the yield of PPV VLPs reached 2.5 g/L, quantified by HPLC, using a defined mineral medium after 48 h fermentation. Two strategies were established to purify intracellular PPV VLPs: (i) Using the cation exchange chromatography coupled with Sephacryl® S-500 HR chromatography to purify VLPs from the supernatants of pH adjusted cell lysates. (ii) Using anion exchange chromatography followed by cross-flow diafiltration to recover the VLPs precipitated in pH adjusted cell lysates. The purity of PPV VLPs reached about 95%, and total recovery was more than 60%. Vaccination of mice with the purified PPV VLPs induced high titers of specific IgG antibodies in sera, and showed hemagglutination inhibitions on both swine and guinea pig erythrocytes. Spleen lymphocyte proliferation and cytokines detection suggested the PPV VLPs produced by K. marxianus provoked the cellular immune and humoral immunity responses in mice.ConclusionsThis is the highest production of recombinant PPV VLPs achieved to date. The superiorities, Generally Recognized As Safe (GRAS), high production, short lead time, and low cost, make K. marxianus a greatly competitive platform for bioproduction of PPV VLPs vaccine.

A stable platform for the production of virus-like particles pseudotyped with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein

In this study, we showed that a codon optimized version of the spike (S) protein of SARS-CoV-2 can migrate to the cell membrane. However, efficient production of Moloney murine leukemia (MLV) infectious viral particles was only achieved with stable expression of a shorter S version in C-terminal (ΔS) in MLV Gag-pol expressing cells. As compared to transient transfections, this platform generated viruses with a 1000-fold higher titer. ΔS was 15-times more efficiently incorporated into VLPs as compared to S, and that was not due to steric interference between the cytoplasmic tail and the MLV capsid, as similar differences were also observed with extracellular vesicles. The amount of ΔS incorporated into VLPs released from producer cells was high and estimated at 1.25 μg/mL S2 equivalent (S is comprised of S1 and S2). The resulting VLPs could potentially be used alone or as a boost of other immunization strategies for COVID-19.

Efficient Production of Chimeric Hepatitis B Virus-Like Particles Bearing an Epitope of Hepatitis E Virus Capsid by Transient Expression in Nicotiana benthamiana.

The core antigen of hepatitis B virus (HBcAg) is capable of self-assembly into virus-like particles (VLPs) when expressed in a number of heterologous systems. Such VLPs are potential carriers of foreign antigenic sequences for vaccine design. In this study, we evaluated the production of chimeric HBcAg VLPs presenting a foreign epitope on their surface, the 551–607 amino acids (aa) immunological epitope of the ORF2 capsid protein of hepatitis E virus. A chimeric construct was made by the insertion of 56 aa into the immunodominant loop of the HBcAg. The sequences encoding the chimera were inserted into the pEAQ-HT vector and infiltrated into Nicotiana benthamiana leaves. The plant-expressed chimeric HBcHEV ORF2 551–607 protein was recognized by an anti-HBcAg mAb and anti-HEV IgG positive swine serum. Electron microscopy showed that plant-produced chimeric protein spontaneously assembled into “knobbly” ~34 nm diameter VLPs. This study shows that HBcAg is a promising carrier platform for the neutralizing epitopes of hepatitis E virus (HEV) and the chimeric HBcAg/HEV VLPs could be a candidate for a bivalent vaccine.

Production of mosquito cell-derived Zika virus-like particles using BacMos system

Background: Zika virus (ZIKV) is a mosquito-borne flavivirus which has been conclusively linked to Guillain-Barré syndrome and microcephaly. The worldwide emergence of ZIKV has greatly increased the demand for vaccines that reduce or prevent disease transmission. Neutralizing human antibodies which target ZIKV E proteins have been shown to prevent ZIKV replication. Virus-like particles (VLPs) lacking viral genetic material comprise self-assembled multi-subunit protein structures that are capable of strongly activating humoral and cellular immunity. Flavivirus prM and E proteins are both necessary and sufficient for the production of VLPs. Thus, it appears that ZIKV VLPs are an ideal target for vaccine design and serological detection. Methods: In this study, the BacMos (baculovirus/mosquito) method was used to introduce the ZIKV prME gene into mosquito cells. Immunofluorescence assays (IFAs), dot blot (DB) analysis, and Western blot (WB) analysis were used to evaluate the expression and secretion of ZIKV glycoproteins. VLP formation was confirmed using transmission electron microscopic (TEM) and dynamic light scattering (DLS) analysis. Results: IFA presented intense signals from ZIKV E-positive cells in BacMos-ZIKV prME-transduced cells. DB and WB detected abundant ZIKV glycoproteins in the culture medium of BacMos-ZIKV prME-transduced cells. TEM observation and DLS analysis revealed that ZIKV VLPs comprised spherical particles, with an average diameter of 30 nm. Conclusions: Mosquito cell-derived ZIKV VLPs are promising candidates for the development of safe, efficacious vaccines and diagnostic antigens in the future.

The MultiBac BEVS: Basics, applications, performance and recent developments.

The baculovirus expression vector system (BEVS) delivers high yield heterologous protein expression and is widely used in academic and industrial R&D. The proteins produced enable many applications including structure/function analysis, drug screening and manufacture of protein therapeutics. Vital cellular functions are controlled by multi-protein complexes, MultiBac, a BEVS specifically designed for heterologous multigene delivery and expression, has unlocked many of these machines to atomic resolution studies. Baculovirus can accommodate very large foreign DNA cargo for faithful delivery into a target host cell, tissue or organism. Engineered MultiBac variants exploit this valuable feature for delivery of customized multifunctional DNA circuitry in mammalian cells and for production of virus-like particles for vaccines manufacture. Here, latest developments and applications of the MultiBac system are reviewed.

Accelerating HIV-1 VLP production using stable High Five insect cell pools.

Stable cell pools are receiving a renewed interest as a potential alternative system to clonal cell lines. The shorter development timelines and the capacity to achieve high product yields make them an interesting approach for recombinant protein production. In this study, stable High Five cell pools are assessed for the production of a simple protein, mCherry, and the more complex HIV-1 Gag-eGFP virus-like particles (VLPs). Random integration coupled to fluorescence-activated cell sorting (FACS) in suspension conditions is applied to accelerate the stable cell pool generation process and enrich it with high producer cells. This methodology is successfully transferred to a bioreactor for VLP production, resulting in a 2-fold increase in VLP yields with respect to shake flask cultures. In these conditions, maximum viable cell concentration improves by 1.5-fold, and by-product formation is significantly reduced. Remarkably, a global increase in the uptake of amino acids in the Gag-eGFP stable cell pool is observed when compared with parental High Five cells, reflecting the additional metabolic burden associated with VLP production. These results suggest that stable High Five cell pools are a robust and powerful approach to produce VLPs and other recombinant proteins, and put the basis for future studies aiming to scale up this system.

Characterization of Zika virus-like particles as potential vaccine antigens

Background: Many of emerging and re-emerging epidemics are caused by viruses belonging to the Flaviviridae family. The family includes, among others, Dengue virus (DENV), West Nile virus, tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV). ZIKV is a mosquito-transmitted flavivirus, which was found to be related to several diseases including infant microcephaly, Guillain-Barre Syndrome and some other neurological defects. In recent years ZIKV has spread throughout Asia, Africa and America and it co-circulates with other flaviviruses. Several laboratories have reported possible antibody-dependent enhancement of DENV infection after previous ZIKV infection. Therefore, the rational approach to the development of an effective vaccine is highly recommended. Currently, many types of potential vaccines are being tested: live-attenuated and inactivated viruses, viral vectors carrying ZIKV genes, viral DNA, mRNA and protein subunits, and virus-like particles (VLPs). This last option–VLPs, are characterized by lack of genetic material, which makes them non-infectious. ZIKV VLPs, similarly to most of other flaviviruses, are composed of viral proteins (prM/M and E), organized almost identically to the native virions. VLPs can be produced in different expression system: insect, mammalian and yeast cells. However, these systems differ in terms of culturing conditions, production efficiency, and post-translational modifications of protein. Methods and materials: Therefore, the main aim of the study was to evaluate ZIKV VLPs produced in two different expression systems, insect and mammalian cells, as vaccine antigens. Production of VLPs was monitored by Western blotting, dynamic light scattering (DLS), sucrose gradient sedimentation as well as by electron microscopy. Functional and conformational analysis of obtained VLPs was performed by enzyme-linked immunosorbent assay (ELISA) using a panel of conformational antibodies. Comparative analysis of immunological properties was performed using mouse model. Results: VLPs in two expression systems were successfully produced. Our study suggests that VLPs produced in various expression systems may differ in protein composition and conformation. All examined ZIKV VLPs were immunogenic as assayed by ELISA but varied greatly in virus neutralization efficacy. Conclusion: Our studies have confirm that ZIKV VLPs offer a promising approach for effective vaccination, even though the expression system greatly influences the antigenic and immunogenic properties.