Virus-like Particles Research Articles

Компоненты микробной пищевой сети в пелагиали фьордов о. Западный Шпицберген в современных климатических условиях

The spatial variability of quantitative indicators of prokaryotes and virus-like particles in the waters of three Arctic fjords was studied in connection with the structure of their dominant water masses in the summer. A comparison of the abundance and biomass of prokaryotes showed that their average value in mesotrophic surface waters (0.60 million cells/ml and 28.64 mg/m3, respectively) exceeded the values in deeper oligotrophic waters – intermediate (0.33 million cells/ml and 16.82 mg/m3) and winter (0.32 million cells/ml and 19.72 mg/m3). The abundance of virus-like particles in different trophic conditions positively correlated with the parameters of prokaryotic communities and in the isolated water masses averaged 1.01 million, 0.09 million and 1.31 million per ml respectively. The factors determining the distribution of the most abundant representatives of the microbial food web and the nature of their interaction are considered.

Phenotypic Shift of an Inflammatory Eosinophil Subset into a Steady-State Resident Phenotype after 2 Years of Vaccination against IL-5 in Equine Insect Bite Hypersensitivity

Eosinophils play a key role in allergic diseases such as insect bite hypersensitivity (IBH). Together with Th2 cells, they shape the course of inflammation in associated type I/IVb allergies. Therefore, a virus-like particle (VLP)-based vaccine targeting equine interleukin-5 (eIL-5), eIL-5-CuMV-TT, was developed to interfere with the IL-5 dependency of eosinophils by inducing the production of anti-self-IL-5 antibodies and alleviating clinical signs in IBH-affected horses. A previous study highlighted the presence of two eosinophil subsets, steady-state resident eosinophils (rEos) and inflammatory eosinophils (iEos), circulating in the blood of healthy and IBH-affected horses, distinguishable by the expression of integrin CD49f. Furthermore, eIL-5-CuMV-TT 1st year vaccination showed a significant decrease of total eosinophils and, in particular, iEos. Nevertheless, the very few remaining eosinophils still shared an iEos phenotype, reflected by bigger size and higher granularity. The aim of this study was to follow up on the phenotype of eosinophils in the 2nd year of vaccination of IBH-affected horses with eIL-5-CuMV-TT. Using flow cytometry analysis of the blood of healthy, IBH, IBH-placebo, and IBH-vaccinated horses, the percentage and count of cells were compared between groups with a focus on pair analysis of eosinophils in 1st and 2nd year vaccinated horses. Our data showed comparably low levels of iEos and a significant increase of rEos in 2nd year compared to 1st year vaccinated horses, suggesting a phenotypic shift toward a resident-like eosinophil population, primarily associated with the phenotype of healthy horses. The reduction of size, granularity, and expression of integrin CD49f in the 2nd year suggests a benefit of long-term treatment with the eIL-5-CuMV-TT vaccine.

Effective removal of host cell-derived nucleic acids bound to hepatitis B core antigen virus-like particles by heparin chromatography.

Virus-like particles (VLPs) show considerable potential for a wide array of therapeutic applications, spanning from vaccines targeting infectious diseases to applications in cancer immunotherapy and drug delivery. In the context of hepatitis B core antigen (HBcAg) VLPs, a promising candidate for gene delivery approaches, the naturally occurring nucleic acid (NA) binding region is commonly utilized for effective binding of various types of therapeutic nucleic acids (NAther). During formation of the HBcAg VLPs, host cell-derived nucleic acids (NAhc) might be associated to the NA binding region, and are thus encapsulated into the VLPs. Following a VLP harvest, the NAhc need to be removed effectively before loading the VLP with NAther. Various techniques reported in literature for this NAhc removal, including enzymatic treatments, alkaline treatment, and lithium chloride precipitation, lack quantitative evidence of sufficient NAhc removal accompanied by a subsequent high VLP protein recovery. In this study, we present a novel heparin chromatography-based process for effective NAhc removal from HBcAg VLPs. Six HBcAg VLP constructs with varying lengths of the NA binding region and diverse NAhc loadings were subjected to evaluation. Process performance was thoroughly examined through NAhc removal and VLP protein recovery analyses. Hereby, reversed phase chromatography combined with UV/Vis spectroscopy, as well as silica spin column-based chromatography coupled with dye-based fluorescence assay were employed. Additionally, alternative process variants, comprising sulfate chromatography and additional nuclease treatments, were investigated. Comparative analyses were conducted with LiCl precipitation and alkaline treatment procedures to ascertain the efficacy of the newly developed chromatography-based methods. Results revealed the superior performance of the heparin chromatography procedure in achieving high NAhc removal and concurrent VLP protein recovery. Furthermore, nuanced relationships between NA binding region length and NAhc removal efficiency were elucidated. Hereby, the construct Cp157 surpassed the other constructs in the heparin process by demonstrating high NAhc removal and VLP protein recovery. Among the other process variants minimal performance variations were observed for the selected constructs Cp157 and Cp183. However, the heparin chromatography-based process consistently outperformed other methods, underscoring its superiority in NAhc removal and VLP protein recovery.

Production of Norovirus VLPs of the Nine Representative Genotypes Widely Distributed in Japan using the Silkworm-Baculovirus Expression Vector System

Norovirus (NoV) is one of the major causes of acute viral gastroenteritis in humans. Genetic variation is abundant, and prevalent genotypes vary from year to year and region to region. Since NoVs are difficult to amplify in cultured cells, genome RNA-free virus-like particles (VLPs) that mimic the capsid structure of the virus are promising vaccine candidates for the prevention of NoVs infection, and the development of multivalent VLP vaccines is required to prevent NoV infection in a wide range of genotypes. In this study, we attempted to produce NoV VLPs of the top nine genotypes that have a history of epidemics in Japan using the silkworm-baculovirus expression vector system (silkworm-BEVS), which has a proven track record in the mass production of recombinant proteins. In silkworm pupae infected with recombinant baculoviruses constructed to express VP1s, the major protein that forms VLP, the NoV VP1 protein was expressed in large amounts. Most genotypes of VP1 accumulated in the cytoplasm as soluble proteins, but solubility was reduced for that of two genotypes. VP1s of five genotypes could be purified in large quantities (>0.9mg per pupa) by a two-step purification process, and gel filtration chromatography analysis confirmed the formation of VLPs. This study demonstrates the utility of silkworm-BEVS in producing NoV VLPs of multiple genotypes and provides the basis for the development of a multivalent vaccine against genetically diverse NoV infections.

Preparation of bovine coronavirus virus-like particles and its immunogenicity in mice and cattle

The widespread prevalence of bovine coronavirus (BCoV) disease worldwide has impacted the livestock industry economically. No effective vaccine is available in China. In this study, we produced BCoV virus-like particles (VLPs) containing E, M, N, S, and hemagglutinin-esterase (HE) proteins using a baculovirus expression system. Five recombinant baculoviruses were co-infected with Sf9 cells, and the VLPs were assembled and characterized. Mice and cattle were immunized by VLPs mixed with MF59 and CpG 55.2 adjuvants. Two doses of the VLPs/MF59/CpG vaccine were administered in mice and cattle. The immune effect of the VLPs/MF59/CpG vaccine was measured using indirect ELISA and neutralization assays. After immunization, the serum IgG-specific antibody titer against S protein and neutralization antibody titer increased to 1:1.28 × 104 (p < 0.01) and 1:128 (p < 0.01) in mice, respectively. Interestingly, the high IgG antibody and neutralizing antibody titers were maintained for seven days in mice. In addition, the serum IgG-specific antibody titer against S proteins and neutralization antibody titer increased to 1:1.024 × 105 and 1:512 (p < 0.05) in cattle, respectively. The high IgG antibody and neutralizing antibody titers were maintained for 21 days in cattle. Notably, BCoV VLPs group interferon-gamma (IFN-γ) lymphocytes in spleens were significantly increased (p < 0.01). These findings suggest that BCoV VLPs induced strong cellular and humoral immune responses in mice and cattle. These findings suggest that BCoV VLPs could serve as a potent immunogen for vaccine development.

Highly pure measles virus generated by combination of salt-active nuclease treatment and heparin affinity chromatography

Highly purified virus preparations are essential for accurate activity and potency determination. This requires simple and efficient purification methods, especially in the early stages of research and development. While heparin affinity chromatography has been already successfully used for the purification of several enveloped viruses and virus-like particles, we extended its use to purification of very sensitive measles virus. The performance of heparin and heparin-like affinity chromatography was evaluated for the purification of recombinant measles virus, a large and labile enveloped virus used as vaccine or cancer therapy. Since DNA, particularly in the form of chromatin is a critical impurity in enveloped virus preparations, the effect of integration of an endonuclease (Benzonase® or M-SAN) treatment prior to chromatography was also investigated.Both, Capto™ DeVirS (heparin-like) and Capto™ Heparin were able to capture measles viruses directly from clarified cell culture supernatant. Despite capturing 100% of infectious measles virus, low recovery (8%) was observed for Capto™ DeVirS. For Capto™ Heparin recoveries up to 85% were observed. The combination of M-SAN with Capto™ Heparin enabled the production of highly purified measles virus with a yield of 62% and a final purity of 10.2 ng dsDNA per dose (1×105), outperforming the processes without endonuclease treatment with a yield of 18%, and a purity of 66.7 ng dsDNA/dose or using Benzonase® with a yield of 38% and a purity of 21.2 ng dsDNA/dose. As the developed method is simple and scalable it could also be integrated in a downstream process train for measles virus manufacturing.

An improved high-resolution method for quantitative separation of empty and filled AAV8 capsids by strong anion exchange HPLC.

Cell and gene therapy (CGT) is a field of therapeutic medicine that aims to treat, prevent, and cure diseases using engineered cells (stem cells, immune cells, and differentiated adult or fetal cells), vectors [Adeno Associated Virus (AAV), Adeno Virus (AV), Herpes Simplex Virus (HSV), Baculo Virus (BV), Lenti Virus (LV), Retro Virus (RV), etc.], and other carriers [non-viral vectors, virus-like particles (VLP), Lipid Nano-Particles (LNP), etc.]. Among viral CGT vectors, adeno-associated viruses and lentiviruses (AAV and LV) are the most widely applied vector platforms. The presence of non-functional (empty or non-infectious) vectors that carry null or partial genes in the final drug product is classified as an impurity by the FDA. These impurities impair dosage accuracy and induce non-specific immunogenicity and variability in drug efficacy. These non-functional viral vectors in the drug product need to be elucidated following International Conference on Harmonization (ICH) guidelines for clinical manufacturing of the final drug product. This article showcases an ion-exchange chromatography (IEX) high-resolution method supporting ICH guidelines using commercially available AAV8 filled and empty capsids as reference standards. Our method successfully separated empty to full capsids with a resolution of 15 and sustained a linearity greater than 0.98 even under a wide range of empty or full viral particle concentrations (E+9 to E+13 vp/mL), which is an upgrade to other IEX capsid separation methods. The medium-throughput capacity and shorter sample processing time improve testing efficiency and save costs while delivering quality as value. The discussed method is a reliable and reproducible platform to precisely evaluate the presence of non-functional viral particles in AAV8 samples. Aligned with other orthogonal results, the method is a powerful tool to improve the quality of rAAV analytics.

Unveiling the Dynamic Mechanism of SARS-CoV-2 Entry Host Cells at the Single-Particle Level.

Understanding the dynamic features of severe acute respiratory coronavirus 2 (SARS-CoV-2) binding to the cell membrane and entry cells is crucial for comprehending viral pathogenesis and transmission and facilitating the development of effective drugs against COVID-19. Herein, we employed atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) to study the binding dynamics between the virus and cell membrane. Our findings revealed that the Omicron variant of SARS-CoV-2 virus-like particles (VLPs) exhibited a slightly stronger affinity for the angiotensin-converting enzyme-2 (ACE2) receptor compared with the Delta variant and was significantly higher than the wild-type (WT). Using a real-time force-tracing technique, we quantified the dynamic parameters for a single SARS-CoV-2 VLP entry into cells, showing that approximately 200 ms and 60 pN are required. The parameters aligned with the analysis obtained from coarse-grained molecular dynamics (CGMD) simulations. Additionally, the Omicron variant invades cells at a higher entry cell speed, smaller force, and higher probability. Furthermore, single-particle fluorescence tracking visually demonstrated clathrin-dependent endocytosis for SARS-CoV-2 entry into A549 cells. The dynamic features of endocytosis provide valuable insights into the SARS-CoV-2 entry mechanism and possible intervention strategies targeting the viral infection process.

Beyond traditional vaccines: Semi-purified low-pathogenic avian influenza H9N2 virus-like particles and their promise for broiler immunity

Background and Aim: Avian influenza is a global threat to avian species, particularly in developing countries. Recombinant vaccines, including virus-like particles (VLPs), are promising strategies for preventing the spread of the disease. VLPs produced through the self-assembly of viral structural proteins without genomic material mimic native virions and are promising platforms for new vaccines. VLPs have been shown to elicit protective antibodies and are effective and safe vaccines against influenza. This study aimed to optimize the protocol for the production and characterization of H9N2 VLPs and their evaluation as a vaccine in broiler birds. Materials and Methods: Low-pathogenic influenza virus (LPAI) H9N2 was isolated and characterized through whole-genome sequencing, and a VLP-based vaccine for LPAI H9N2 was prepared using a baculovirus expression system. Codon-optimized hemagglutinin (HA), neuraminidase (NA), and M1 were successfully cloned in pFastbac1 and expressed in SF9 cells. Proteins were characterized using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and electron microscopy after purification. Semi-purified proteins were tested as a vaccine in broiler chickens challenged with LPAI H9N2. Results: Recombinant Bacmid DNA from positive clones was extracted and confirmed using a polymerase chain reaction. The transfection showed cytopathic effects, and the proteins were confirmed through western blotting and SDS-PAGE, which showed the sizes of HA = 62–64 KD, NA = 52 KD, and M1 = 25 KD. The shape and morphology were confirmed through transmission electron microscopy which revealed 100–150 nm size particles. As a result, the semi-purified VLPs (HA assay: 256) were tested as a vaccine for specific-pathogen free broiler birds; administered through subcutaneous and intranasal routes. The birds were challenged on the 28th day after vaccination with the H9N2 strain, and the birds showed significant cross-reactivity with the H9N2 strain. Conclusion: The semi-purified VLP-based vaccine induced a significant immune response in vivo. This vaccine formulation has the potential to control avian influenza outbreaks in Pakistan’s poultry industry. Keywords: avian influenza virus, baculovirus expression system, H9N2, recombinant vaccine, virus-like particles.

Broadly cross-reactive immune responses in chickens immunized with chimeric virus-like particles of nodavirus displaying the M2e originated from avian and human influenza A viruses

Avian influenza A viruses (IAVs) pose a persistent threat to poultry industry worldwide, despite the presence of vaccines. Additionally, reverse-zoonosis transmission potentially introduces human-originated IAVs into poultry and complicates the efforts to control the spread of influenza. Current avian influenza vaccines are primarily based upon the rapidly mutating hemagglutinin (HA) and neuraminidase (NA) glycoproteins, which limit their efficacy against diverse strains of IAVs. Hence, the highly conserved ectodomains of matrix 2 protein (M2e) of IAVs are widely studied as alternatives to the HA and NA. However, the differences in the M2e amino acid sequences between avian and human IAVs generate antibodies that do not cross-react reciprocally with IAVs from other origins. To broaden and enhance the immunogenicity of M2e, we fused two copies each of the M2e derived from avian and human IAVs at the C-terminal end of the Macrobrachium rosenbergii nodavirus (MrNV) capsid protein (NvC). Transmission electron microscopic and dynamic light scattering analyses revealed that the chimeric protein self-assembled into virus-like particles (VLPs). Immunization of chickens with the chimeric VLPs demonstrated a robust induction of broadly reactive immune responses against both the M2e of avian and human IAVs. Additionally, the chimeric VLPs elicited the production of cytotoxic T lymphocytes (CTL), macrophages, as well as a well-balanced Th1 and Th2 population, indicating their potential in activating cell-mediated immune responses in chickens. Furthermore, the chimeric VLPs triggered the production of both Th1- and Th2-cytokines, attesting their potential in mounting a robust and balanced immune response in avian species. This study demonstrated the potential of these chimeric VLPs in stimulating and broadening cross-reactive immune responses in chickens against both avian and human IAVs.

Ebola Virus Matrix Protein VP40 Single Mutations G198R and G201R Significantly Enhance Plasma Membrane Localization.

Viral proteins frequently undergo single or multiple amino acid mutations during replication, which can significantly alter their functionality. The Ebola virus matrix protein VP40 is multifunctional but primarily responsible for creating the viral envelope by binding to the inner leaflet of the host cell plasma membrane (PM). Changes to the VP40 surface cationic charge via mutations can influence PM interactions, resulting in altered viral assembly and budding. A recent mutagenesis study evaluated the effects of several mutations and found that mutations G198R and G201R enhanced VP40 assembly at the PM and virus-like particle budding. These two mutations lie in the loop region of the C-terminal domain (CTD), which directly interacts with the PM. To understand the role of these mutations in PM localization at the molecular level, we performed both all-atom and coarse-grained molecular dynamics simulations using a dimer-dimer configuration of VP40, which contains the CTD-CTD interface. Our studies indicate that the location of mutations on the outer surface of the CTD regions can lead to changes in membrane binding orientation and degree of membrane penetration. Direct PI(4,5)P2 interactions with the mutated residues seem to further stabilize and pull VP40 into the PM, thereby enhancing interactions with numerous amino acids that were otherwise infrequently or completely inaccessible. These multiscale computational studies provide new insights at the atomic and molecular level as to how VP40-PM interactions are altered through single amino acid mutations. Given the high case fatality rates associated with Ebola virus disease in humans, it is essential to explore the mechanisms of viral assembly in the presence of mutations to mitigate the severity of the disease and understand the potential of future outbreaks.

Highly stable Saccharomyces cerevisiae L-BC capsids with versatile packing potential.

Virus-like particles (VLPs) are promising nanoscaffolds in development of vaccines and nanodelivery systems. Along with efficient production in various expression systems, they also offer extensive functionalization options. Nevertheless, the ultimate integrity of VLPs is an important burden for the applicability in nanobiotechnology. In this study, we characterize the Saccharomyces cerevisiae L-BC VLPs synthesized and purified from Escherichia coli and Saccharomyces cerevisiae cells. The particles exhibited prominent size stability in buffers within a range of ionic strength conditions, pH environment and presence of magnesium ions during the long-term storage at temperatures up to 37°C. Bacteria-derived particles exhibited alleviated stability in acidic pH values, higher ionic strength and temperature compared to yeast-derived particles. Taking advantage of gene engineering, 120 copies of red fluorescent protein mCherry were successfully encapsulated into both preparations of L-BC VLPs, while passive diffusion enabled encapsulation of antimicrobial peptide nisin into the yeast-derived unmodified VLPs. Our findings indicate that L-BC VLPs generally exhibit high long-term stability under various conditions, while yeast-derived L-BC VLPs are more stable under the elevated temperatures than bacteria-derived particles. Stability studies and encapsulation of particles by different molecules involving alternative strategies delineate the L-BC VLP potential to be developed into versatile nanodelivery system.

Packaging of alphavirus-based self-amplifying mRNA yields replication-competent virus through a mechanism of aberrant homologous RNA recombination.

Messenger (m)RNA has taken center stage in vaccine development, gene therapy, and cancer immunotherapy. A next-generation of mRNA is the self-amplifying (sa)mRNA, which induces broad and long-lasting immunity at a lower dose which provides better clinical outcomes in conjunction with fewer adverse effects. SamRNA, also known as "replicon" RNA, encodes the replication machinery of an alphavirus together with an antigen. Efficient delivery of replicon RNA to target tissues can be accomplished by packaging the replicon RNA in virus-like replicon particles (VRPs) via co-transfection of producer cells with defective helper RNA(s) encoding the alphavirus structural proteins. During the manufacture of VRPs, however, there is a potential risk of RNA recombination, which may lead to the formation of replication-competent virus (RCV). To investigate the factors influencing the unwanted RCV formation, we evaluated how sequence homology orchestrates alphavirus RNA recombination. Several combinations of complementing alphavirus replicon and helper RNAs varying in length of sequences overlap were co-transfected in mammalian cells. The culture fluid was serially passaged to detect RCV. Nanopore sequencing of cells after the first passage in combination with amplicon-based Sanger sequencing of RCV in the culture fluid after four passages led to the detection of RNA recombination. RCV was generated between replicon and helper RNAs with sequence homology in either the non-structural or structural genes, whereas RNAs without overlapping gene regions did not generate RCV. Remarkably, no sequence overlap was detected at the recombination junction sites in the RCV genome, suggesting a mechanism of "aberrant homologous RNA recombination." Accordingly, we conclude that the alphavirus RNA recombination process leading to the formation of RCV is homology-assisted and can be prevented by avoiding sequence homology between replicon and helper RNAs.IMPORTANCEThere is a growing interest in the use of self-amplifying (sa)mRNA vectors for next-generation vaccine development, gene therapy, and cancer immunotherapy. The delivery of samRNA in the form of virus-like replicon particles (VRPs) enables efficient delivery of samRNA to target tissue. The production of these VRPs, however, suffers from contamination with replication-competent virus (RCV) that is thought to arise from recombination events between samRNA and helper RNAs for VRP packaging. The presence of RCV in samRNA in the clinical product is undesirable as alphaviruses may cause serious disease in humans. However, the underlying recombination mechanism leading to RCV is currently unknown. In our work, we demonstrate a detailed evaluation of the recombination sites, which indicates that RCV is formed through an unusual mechanism of "aberrant homologous RNA recombination." The results are useful for researchers in the field of RNA vaccine manufacture and delivery.

Heterologous immunization targeting the CST1 antigen confers better protection than ROP18 in mice.

Aim: To evaluate the protective efficacy induced by heterologous immunization with recombinant baculoviruses or virus-like particles targeting the CST1 and ROP18 antigens of Toxoplasma gondii.Materials & methods: Recombinant baculovirus and virus-like particle vaccines expressing T. gondii CST1 or ROP18 antigens were developed to evaluate protective immunity in mice upon challenge infection with 450 Toxoplasma gondii (ME49).Results: Immunization with CST1 or ROP18 vaccines induced similar levels of T. gondii-specific IgG and IgA responses. Compared with ROP 18, CST1 vaccine showed better antibody-secreting cell response, germinal center B cell activation, and significantly reduced brain cyst burden and body weight loss.Conclusion: Our findings suggest that CST1 heterologous immunization elicited better protection than ROP18, providing important insight into improving the toxoplasmosis vaccine design strategy.

Plant-based vaccines against viral hepatitis: A panoptic review.

The traditional vaccines against hepatitis have been instrumental in reducing the incidence of some types of viral hepatitis; however, the need for cost-effective, easily distributable, and needle-free vaccine alternatives has led to the exploration of plant-based vaccines. Plant-based techniques offer a promising avenue for producing viral hepatitis vaccines due to their low-cost cultivation, scalability, and the potential for oral administration. This review highlights the successful expression of hepatitis B surface antigens in plants and the subsequent formation of virus-like particles, which have shown immunogenicity in preclinical and clinical trials. The challenges such as achieving sufficient antigen expression levels, ensuring consistent dosing, and navigating regulatory frameworks, are addressed. The review considers the potential of plant-based vaccines to meet the demands of rapid vaccine deployment in response to outbreaks and their role in global immunization strategies, particularly in resource-limited settings. This review underscores the significant strides made in plant molecular farming and the potential of plant-based vaccines to complement existing immunization methods against viral hepatitis.

Chemometrics and analytical blank on the at-line monitoring of Zika-VLP production using near-infrared spectroscopy

The Zika disease caused by the Zika virus was declared a Public Health Emergency by the World Health Union (WHO), with microcephaly as the most critical consequence. Aiming to reduce the spread of the virus, biopharmaceutical organizations invest in vaccine research and production, based on multiple platforms. A crescent vaccine production approach is based on virus-like particles (VLP), for not having genetic material in its composition, hypoallergenic and non-mutant character. For bioprocess, it is essential to have means of real-time monitoring, which can be assessed using process analysis techniques such as Near-infrared (NIR) spectroscopy, that can be combined with chemometric methods, like Partial-Least Squares (PLS) and Artificial Neural Networks (ANN) for prediction of biochemical variables. This work proposes a biochemical Zika VLP upstream production at-line monitoring model using NIR spectroscopy comparing sampling conditions (with or without cells), analytical blank (air, ultrapure water), and spectra pre-processing approaches. Seven experiments in a benchtop bioreactor using recombinant baculovirus/Sf9 insect cell platform in serum-free medium were performed to obtain biochemical and spectral data for chemometrics modeling (PLS and ANN), composed by a random data split (80 % calibration, 20 % validation) for cross-validation of the PLS models and 70 % training, 15 % testing, 15 % validation for ANN. The best models generated in the present work presented an average absolute error of 1.59 × 105 cell/mL for density of viable cells, 2.37 % for cell viability, 0.25 g/L for glucose, 0.007 g/L for lactate, 0.138 g/L for glutamine, 0.18 g/L for glutamate, 0,003 g/L for ammonium, and 0.014 g/L for potassium.

Vaccine Potency and Structure of Yeast-Produced Polio Type 2 Stabilized Virus-like Particles.

Poliovirus (PV) is on the brink of eradication due to global vaccination programs utilizing live-attenuated oral and inactivated polio vaccines. Recombinant PV virus-like particles (VLPs) are emerging as a safe next-generation vaccine candidate for the impending polio-free era. In this study, we investigate the production, antigenicity, thermostability, immunogenicity, and structures of VLPs derived from PV serotype 2 (PV2) wildtype strain and thermally stabilized mutant (wtVLP and sVLP, respectively). Both PV2 wtVLP and sVLP are efficiently produced in Pichia pastoris yeast. The PV2 sVLP displays higher levels of D-antigen and significantly enhanced thermostability than the wtVLP. Unlike the wtVLP, the sVLP elicits neutralizing antibodies in mice at levels comparable to those induced by inactivated polio vaccine. The addition of an aluminum hydroxide adjuvant to sVLP results in faster induction and a higher magnitude of neutralizing antibodies. Furthermore, our cryo-EM structural study of both sVLP and wtVLP reveals a native conformation for the sVLP and a non-native expanded conformation for the wtVLP. Our work not only validates the yeast-produced PV2 sVLP as a promising vaccine candidate with high production potential but also sheds light on the structural mechanisms that underpin the assembly and immunogenicity of the PV2 sVLP. These findings may expedite the development of sVLP-based PV vaccines.

A single dose of an ALVAC vector-based RABV virus-like particle candidate vaccine induces a potent immune response in mice, cats and dogs

Rabies, caused by the Rabies virus (RABV), is a highly fatal zoonotic disease. Existing rabies vaccines have demonstrated good immune efficacy, but the complexity of immunization procedures and high cost has impeded the elimination of RABV, particularly in the post-COVID-19 era. There is a pressing need for safer and more effective rabies vaccines that streamline vaccination protocols and reduce expense. To meet this need, we have developed a potential rabies vaccine candidate called ALVAC-RABV-VLP, utilizing CRISPR/Cas9 gene editing technology. This vaccine employs a canarypox virus vector (ALVAC) to generate RABV virus-like particles (VLPs). In mice, a single dose of ALVAC-RABV-VLP effectively activated dendritic cells (DCs), follicular helper T cells (Tfh), and the germinal center (GC)/plasma cell axis, resulting in durable and effective humoral immune responses. The survival rate of mice challenged with lethal RABV was 100%. Similarly, in dogs and cats, a single immunization with ALVAC-RABV-VLP elicited a stronger and longer-lasting antibody response. ALVAC-RABV-VLP induced superior cellular and humoral immunity in both mice and beagles compared to the commercial inactivated rabies vaccine. In conclusion, ALVAC-RABV-VLP induced robust protective immune responses in mice, dogs and cats, offering a novel, cost-effective, efficient, and promising approach for herd prevention of rabies.

Chikungunya Virus Vaccines: A Review of IXCHIQ and PXVX0317 from Pre-Clinical Evaluation to Licensure.

Chikungunya virus is an emerging mosquito-borne alphavirus that causes febrile illness and arthritic disease. Chikungunya virus is endemic in 110 countries and the World Health Organization estimates that it has caused more than 2 million cases of crippling acute and chronic arthritis globally since it re-emerged in 2005. Chikungunya virus outbreaks have occurred in Africa, Asia, Indian Ocean islands, South Pacific islands, Europe, and the Americas. Until recently, no specific countermeasures to prevent or treat chikungunya disease were available. To address this need, multiple vaccines are in human trials. These vaccines use messenger RNA-lipid nanoparticles, inactivated virus, and viral vector approaches, with a live-attenuated vaccine VLA1553 and a virus-like particle PXVX0317 in phase III testing. In November 2023, the US Food and Drug Administration (FDA)approved the VLA1553 live-attenuated vaccine, which is marketed as IXCHIQ. In June 2024, Health Canada approved IXCHIQ, and in July 2024, IXCHIQ was approved by the European Commission. On August 13, 2024, the US FDA granted priority review for PXVX0317. The European Medicine Agency is considering accelerated assessment review of PXVX0317, with potential for approval by both agencies in 2025.In this review, we summarize published data from pre-clinical and clinical trials for the IXCHIQ and PXVX0317 vaccines. We also discuss unanswered questions including potential impacts of pre-existing chikungunya virus immunity on vaccine safety and immunogenicity, whether long-term immunity can be achieved, safety in children, pregnant, and immunocompromised individuals, and vaccine efficacy in people with previous exposure to other emerging alphaviruses in addition to chikungunya virus.

Palmitoylation of SARS-CoV-2 Envelope protein is central to virus particle formation.

The Envelope (E) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an integral structural protein in the virus particles. However, its role in the assembly of virions and the underlying molecular mechanisms are yet to be elucidated, including whether the function of E protein is regulated by post-translational modifications. In the present study, we report that SARS-CoV-2 E protein is palmitoylated at C40, C43, and C44 by palmitoyltransferases zDHHC3, 6, 12, 15, and 20. Mutating these three cysteines to serines (C40/43/44S) reduced the stability of E protein, decreased the interaction of E with structural proteins Spike, Membrane, and Nucleocapsid, and thereby inhibited the production of virus-like particles (VLPs) and VLP-mediated luciferase transcriptional delivery. Specifically, the C40/43/44S mutation of E protein reduced the density of VLPs. Collectively, these results demonstrate that palmitoylation of E protein is vital for its function in the assembly of SARS-CoV-2 particles.IMPORTANCEIn this study, we systematically examined the biochemistry of palmitoylation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) E protein and demonstrated that palmitoylation of SARS-CoV-2 E protein is required for virus-like particle (VLP) production and maintaining normal particle density. These results suggest that palmitoylated E protein is central for proper morphogenesis of SARS-CoV-2 VLPs in densities required for viral infectivity. This study presents a significant advancement in the understanding of how palmitoylation of viral proteins is vital for assembling SARS-CoV-2 particles and supports that palmitoyl acyltransferases can be potential therapeutic targets for the development of SARS-CoV-2 inhibitors.