Virus-like Particles Research Articles

Design of hepadnavirus core protein-based chimeric virus-like particles carrying epitopes from respiratory syncytial virus

Respiratory syncytial virus (RSV) is one of the most important pathogens causing respiratory tract infection in humans, especially in infants and the elderly. The identification and structural resolution of the potent neutralizing epitopes on RSV fusion (F) protein enable an “epitope-focused” vaccine design. However, the display of RSV F epitope II on the surface of the widely-used human hepatitis B virus core antigen (HBcAg) has failed to induce neutralizing antibody response in mice. Here, we used the hepadnavirus core protein (HcAg) from different mammalian hosts as scaffolds to construct chimeric virus-like particles (VLPs) presenting the RSV F epitope II. Mouse immunization showed that different HcAg-based chimeric VLPs elicited significantly different neutralizing antibody responses, among which the HcAg derived from roundleaf bat (RBHcAg) is the most immunogenic. Furthermore, RBHcAg was used as the scaffold platform to present multiple RSV F epitopes, and the immunogenicity was further improved in comparison to that displaying a single epitope II. The designed RBHcAg-based multiple-epitope-presenting VLP formulated with MF59-like adjuvant elicited a potent and balanced Th1/Th2 immune response, and offered substantial protection in mice against the challenge of live RSV A2 virus. The designed chimeric VLPs may serve as the potential starting point for developing epitope-focused vaccines against RSV. Our study also demonstrated that RBHcAg is an effective VLP carrier for presenting foreign epitopes, providing a promising platform for epitope-focused vaccine design.

Antiproliferative and immunomodulative potential of Citrullus colocynthis and its bioactive compounds in human lymphocytes and lung cells

Ethnopharmacological relevanceCitrullus colocynthis (L.) Schrad is a member of the Cucurbitaceae plant family which has been used in traditional medicine for the treatment of lung diseases such as asthma and bronchitis. Aim of the studyThe study was conducted to investigate antiproliferative and immunomodulating effects of C. colocynthis and isolated cucurbitacins on human T lymphocytes and lung epithelial cells in order to evaluate their potential in the treatment of airway diseases. Materials and methodsDifferent concentrations of an ethanolic extract of C. colocynthis fruits and cucurbitacins B (CuB), E (CuE) and E-glucopyranoside (CuE-Glu) were analysed for their cytotoxicity and immunomodulatory potential on Peripheral Blood Mononuclear Cells (PBMCs) of healthy donors and on the epithelial lung cancer cell line A549. Viability and proliferation were tested using WST1 and CFSE assays. Flow cytometric analysis of AnnexinV/PI staining was used to investigate cell death through apoptosis/necrosis. Effects on regulatory mechanisms of T lymphocytes, such as CD69 and CD25 marker activation, cytokine production of the cytokines interleukin 2 (IL2), tumor necrosis factor α (TNFα) and interferon γ (IFNy) were also analysed via flow cytometry. Influences on the activator protein 1 (AP1), nuclear factor of activated T-cells (NFAT) or nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NFκB) pathways were analysed in the Jurkat reporter cell line. Cytokine secretion in A549 cells stimulated with virus-like particles was analysed using the bead-based Legendplex™ assay. ResultsNon-toxic concentrations of C. colocynthis and CuE-Glu showed dose-dependent effects on viability and proliferation in both T lymphocytes and A549 cells. The extracts inhibited lymphocyte activation and suppressed T cell effector functions, which was also shown by lower production of cytokines IL2, TNFα and IFNy. A dose dependent inhibition of the pathways NFκB, NFAT and AP1 in Jurkat cells could be observed. In A549 cells, especially CuE and CuE-Glu showed inhibitory effects on cytokine production following a simulated viral infection. Unglycosylated cucurbitacins were more effective in suppressing the immune function in lymphocytes than glycosylated cucurbitacins, however this activity is limited to cytotoxic concentrations. ConclusionIn our study we could confirm the immunmodulating effect of C. colocynthis and cucurbitacins B, E and E-glucopyranoside in vitro by suppression of different pathways of inflammation and T cell proliferation. Activity in a lung cell model using a virus-like stimulation shows promise for further research regarding cucurbitacins in airway diseases.

Enhanced assembly of bacteriophage T7 produced in cell-free reactions under simulated microgravity

On-demand biomanufacturing has the potential to improve healthcare and self-sufficiency during space missions. Cell-free transcription and translation reactions combined with DNA blueprints can produce promising therapeutics like bacteriophages and virus-like particles. However, how space conditions affect the synthesis and self-assembly of such complex multi-protein structures is unknown. Here, we characterize the cell-free production of infectious bacteriophage T7 virions under simulated microgravity. Rotation in a 2D-clinostat increased the number of infectious particles compared to static controls. Quantitative analyses by mass spectrometry, immuno-dot-blot and real-time PCR showed no significant differences in protein and DNA contents, suggesting enhanced self-assembly of T7 phages in simulated microgravity. While the effects of genuine space conditions on the cell-free synthesis and assembly of bacteriophages remain to be investigated, our findings support the vision of a cell-free synthesis-enabled “astropharmacy”.

Development of a technique for determining the viability of dormant organisms in powdered substances

The purposes of pharmaceutical biotechnology and quality control of probiotics containing lyophilized bacterial preparations, as well as the purpose of ensuring biological safety, require methods for rapid determination of the viability of dormant forms. This paper describes an approach to determining the viability of dry powdered substances. Lyophilized powder of Kalanchoe daigermontiana, dry dormant eggs of Artemia salina, and dry powder of Lycopodium sp. spores were proposed as model objects. Suspensions of virus-like particles (VLPs) to SARS-CoV-2 were proposed as a cell-free reference material. The following methods of elemental analysis were used in the study: X-ray fluorescence spectroscopy (XRF) on an energy-dispersive device, atomic absorption spectroscopy with electrothermal atomization, and Zeeman background correction (GZ-AAS). The free water content was determined by NMR spin echo. A new method was also used for recording the kinetics of changes in broadband radio emission in the centimeter and millimeter wavelength ranges, with a measurement duration of up to 20 minutes. Live and inactivated powdered preparations of K. daigermontiana and A. salina were prepared and characterized by elemental composition, water content, and spin-spin relaxation time (T2). For these live and inactivated preparations, it was determined that the flux density of intrinsic thermal radio emission decreases by an order of magnitude with the loss of viability. In the future, the results obtained will allow for a rapid test of the viability of lyophilized therapeutics (from medicinal plants to bacterial mixtures that stabilize the intestinal microflora).
 Keywords: millimeter emission, X-ray fluorescence analysis, NMR spin echo, nanoparticles, powdered substances, medicinal plant raw materials

Nucleic Acid Vaccines Encoding Proteins and Virus-like Particles for HIV Prevention.

The development of HIV prophylactic vaccines is facing an impasse, since all phase IIb/III clinical trials were halted in 2023 without demonstrating efficacy. Thus, the field is in need of developing novel immunogens and vaccination strategies that induce broadly neutralising antibodies together with potent Fc-dependent effector functions, as well as protective cross-reactive CD4+ and CD8+ T cell responses. Nucleic acid vaccines, particularly mRNA vaccines, have been one of the major groundbreaking advances in the current decade. Nucleic acid vaccines may help recalibrate the HIV vaccine field towards the use of delivery systems that allow the proper expression of immunogens as a sole antigen (i.e., membrane-bound trimeric envelope glycoproteins) or even to be displayed in a multiantigen platform that will be synthesised by the host. In this review, we will summarise how the multiple HIV vaccine strategies pursued in the last 40 years of HIV research have driven current vaccine development, which are the most relevant immunogens identified so far to induce balanced adaptive immune responses, and how they can benefit from the acceptance of nucleic acid vaccines in the market by reducing the limitations of previous delivery systems. The incorporation of nucleic acid vaccines into the current heterogeneous repertoire of vaccine platforms may represent an invaluable opportunity to reignite the fight against HIV.

Interior modification of Macrobrachium rosenbergii nodavirus-like particle enhances encapsulation of VP37-dsRNA against shrimp white spot syndrome infection

BackgroundApplication of a virus-like particle (VLP) as a nanocontainer to encapsulate double stranded (ds)RNA to control viral infection in shrimp aquaculture has been extensively reported. In this study, we aimed at improving VLP’s encapsulation efficiency which should lead to a superior fighting weapon with disastrous viruses.ResultsWe constructed 2 variants of chimeric Macrobrachium rosenbergii nodavirus (MrNV)-like particles (V1- and V2-MrN-VLPs) and tested their efficiency to encapsulate VP37 double stranded RNA as well as WSSV protection in P. vannamei. Two types of short peptides, RNA-binding domain (RBD) and deca-arginine (10R) were successfully engineered into the interior surface of VLP, the site where the contact with VP37-dsRNA occurs. TEM and dynamic light scattering (DLS) analyses revealed that the chimeric VLPs remained their assembling property to be an icosahedral symmetric particle with a diameter of about 30 nm, similar to the original MrN-VLP particle. The superior encapsulation efficiency of VP37-dsRNA into V2-MrN-VLP was achieved, which was slightly better than that of V1-MrN-VLP but far better (1.4-fold) than its parental V0-MrN-VLP which the mole ratio of 7.5–10.5 for all VLP variants. The protection effect against challenging WSSV (as gauged from the level of VP37 gene and the remaining viral copy number in shrimp) was significantly improved in both V1- and V2-MrN-VLP compared with an original V0-MrN-VLP template.ConclusionMrN-VLP (V0-) were re-engineered interiorly with RBD (V1-) and 10R (V2-) peptides which had an improved VP37-dsRNA encapsulation capability. The protection effect against WSSV infection through shrimp administration with dsRNA + V1-/V2-MrN VLPs was experimentally evident.

Virus-like particles of louping ill virus elicit potent neutralizing antibodies targeting multimers of viral envelope protein

Louping ill virus (LIV) is a tick-borne flavivirus that predominantly causes disease in livestock, especially sheep in the British Isles. A preventive vaccine, previously approved for veterinary use but now discontinued, was based on an inactivated whole virion that likely provided protection by induction of neutralizing antibodies recognizing the viral envelope (E) protein. A major disadvantage of the inactivated vaccine was the need for high containment facilities for the propagation of infectious virus, as mandated by the hazard group 3 status of the virus. This study aimed to develop high-efficacy non-infectious protein-based vaccine candidates. Specifically, soluble envelope protein (sE), and virus-like particles (VLPs), comprised of the precursor of membrane and envelope proteins, were generated, characterized, and studied for their immunogenicity in mice. Results showed that the VLPs induced more potent virus neutralizing response compared to sE, even though the total anti-envelope IgG content induced by the two antigens was similar. Depletion of anti-monomeric E protein antibodies from mouse immune sera suggested that the neutralizing antibodies elicited by the VLPs targeted epitopes spanning the highly organized structure of multimer of the E protein, whereas the antibody response induced by sE focused on E monomers. Thus, our results indicate that VLPs represent a promising LIV vaccine candidate.

Learning structural heterogeneity from cryo-electron sub-tomograms with tomoDRGN.

Cryo-electron tomography (cryo-ET) enables observation of macromolecular complexes in their native, spatially contextualized cellular environment. Cryo-ET processing software to visualize such complexes at nanometer resolution via iterative alignment and averaging are well developed but rely upon assumptions of structural homogeneity among the complexes of interest. Recently developed tools allow for some assessment of structural diversity but have limited capacity to represent highly heterogeneous structures, including those undergoing continuous conformational changes. Here we extend the highly expressive cryoDRGN (Deep Reconstructing Generative Networks) deep learning architecture, originally created for single-particle cryo-electron microscopy analysis, to cryo-ET. Our new tool, tomoDRGN, learns a continuous low-dimensional representation of structural heterogeneity in cryo-ET datasets while also learning to reconstruct heterogeneous structural ensembles supported by the underlying data. Using simulated and experimental data, we describe and benchmark architectural choices within tomoDRGN that are uniquely necessitated and enabled by cryo-ET. We additionally illustrate tomoDRGN's efficacy in analyzing diverse datasets, using it to reveal high-level organization of human immunodeficiency virus (HIV) capsid complexes assembled in virus-like particles and to resolve extensive structural heterogeneity among ribosomes imaged in situ.

Generation of a virus-like particles based vaccine against IgE.

Anti-IgE immunotherapy with monoclonal antibodies represents a breakthrough in treatment of severe allergic diseases. However, drawbacks such as short half-life and high price are not negligible. Our objective is to develop an anti-IgE vaccine based on virus-like particles (VLPs) which can induce long-lasting neutralizing IgG anti-IgE antibodies reducing allergic responses without causing intrinsic mast cell activation due to IgE cross-linking. The vaccines were made by chemically coupling three synthetic mouse IgE-Fc fragments to plant-derived immunologically optimized CuMVTT VLPs. The immunogenicity of the vaccines was tested by immunizing naive or allergic mice either with the coupled vaccines or the VLP control followed by systemic or local allergen challenge. Mice immunized with the vaccines exhibited high titers of anti-IgE antibodies in the sera and high levels of anti-IgE secreting plasma cells in lymphoid organs. Moreover, free IgE in serum were reduced by the induced anti-IgE antibodies; therefore, less IgE was bound to FcεRI on the surface of basophils. In line with these reduced IgE levels on effector cells after vaccination, immunized mice were protected from challenge with allergens. Importantly, despite presence of anti-IgE antibodies, no signs of acute or chronic allergic response were seen in immunized allergic mice. The generated vaccines can effectively induce anti-IgE antibodies that did not cause allergic responses in sensitized mice but were able to decrease the level of free and cell bound IgE and protected sensitized animals from allergic responses upon allergen challenge.

Effective and targeted latency reversal in CD4+ T cells from individuals on long term combined antiretroviral therapy initiated during chronic HIV-1 infection

ABSTRACT To date, an affordable, effective treatment for an HIV-1 cure remains only a concept with most “latency reversal” agents (LRAs) lacking specificity for the latent HIV-1 reservoir and failing in early clinical trials. We assessed HIV-1 latency reversal using a multivalent HIV-1-derived virus-like particle (HLP) to treat samples from 32 people living with HIV-1 (PLWH) in Uganda, US and Canada who initiated combined antiretroviral therapy (cART) during chronic infection. Even after 5–20 years on stable cART, HLP could target CD4+ T cells harbouring latent HIV-1 reservoir resulting in 100-fold more HIV-1 release into culture supernatant than by common recall antigens, and 1000-fold more than by chemotherapeutic LRAs. HLP induced release of a divergent and replication-competent HIV-1 population from PLWH on cART. These findings suggest HLP provides a targeted approach to reactivate the majority of latent HIV-1 proviruses among individuals infected with HIV-1.

Structural Relationships to Efficacy for Prazole-Derived Antivirals.

Here, an in vitro characterization of a family of prazole derivatives that covalently bind to the C73 site on Tsg101 and assay their ability to inhibit viral particle production is presented. Structurally, increased steric bulk on the 4-pyridyl of the prazole expands the prazole site on the UEV domain toward the β-hairpin in the Ub-binding site and is coupled to increased inhibition of virus-like particle production in HIV-1. Increased bulk also increased toxicity, which is alleviated by increasing flexibility. Further, the formation of a novel secondary Tsg101 adduct for several of the tested compounds and the commercial drug lansoprazole. The secondary adduct involved the loss of the 4-pyridyl substituent to form an irreversible species, with implications for increasing the half-life of the active species or its specificity toward Tsg101 UEV. It is also determined that sulfide derivatives display effective viral inhibition, presumably through cellular sulfoxidation, allowing for delayed conversion within the cellular environment, and identify SARS-COV-2 as a target of prazole inhibition. These results open multiple avenues for the design of prazole derivatives for antiviral applications.

SARS-CoV-2 virus-like particle variants alpha and delta mimic the native viruses in their differential inflammasome activating potential

The emerging SARS-CoV-2 variants are evolving to evade human immunity and differ in their pathogenicity. While evasion of the variants from adaptive immunity is widely investigated, there is a paucity of knowledge about their interactions with innate immunity. Inflammasome assembly is one of the most potent mechanisms of the early innate response to viruses, but when it is inappropriate, it can perpetuate tissue damage. In this study, we focused on the capacity of SARS-CoV-2 Alpha and Delta variants to activate the NLRP3 inflammasome. We compared the macrophage activation, particularly the inflammasome formation, using Alpha- and Delta-spike virus-like particles (VLPs). We found that VLPs of both variants activated the inflammasome even without a priming step. Delta-spike VLPs had a significantly stronger effect on triggering pyroptosis and inflammasome assembly in THP-1 macrophages than did Alfa-spike VLPs. Cells treated with Delta VLPs showed greater cleavage of caspase-1 and IL-1β release. Furthermore, Delta VLPs induced stronger cytokine secretion from macrophages and caused essential impairment of mitochondrial respiration in comparison to Alpha VLPs. Additionally, infection of primary human monocyte-derived macrophages with the SARS-CoV-2 variants confirmed the observations in VLPs. Collectively, we revealed that SARS-CoV-2 Delta had a greater impact on the inflammasome activation, cell death and mitochondrial respiration in macrophages than did the Alpha variant. Importantly, the differential response to the SARS-CoV-2 variants can influence the efficacy of therapies targeting the host's innate immunity.

Toward a SARS-CoV-2 VLP Vaccine: HBc/G as a Carrier for SARS-CoV-2 Spike RBM and Nucleocapsid Protein-Derived Peptides.

Virus-like particles (VLPs) offer an attractive possibility for the development of vaccines. Recombinant core antigen (HBc) of Hepatitis B virus (HBV) was expressed in different systems, and the E. coli expression system was shown to be effective for the production of HBc VLPs. Here, we used HBc of the HBV genotype G (HBc/G) as a technologically promising VLP carrier for the presentation of spike RBM and nucleocapsid protein-derived peptides of the SARS-CoV-2 Delta variant for subsequent immunological evaluations of obtained fusion proteins. The major immunodominant region (MIR) of the HBc/G protein was modified through the insertion of a receptor binding motif (RBM) from the S protein or B-cell epitope-containing peptide from the N protein. The C-terminus of the two truncated HBc/G proteins was used for the insertion of a group of five cytotoxic T lymphocyte (CTL) epitopes from the N protein. After expression in E. coli, the MIR-derived proteins were found to be insoluble and were recovered through step-wise solubilization with urea, followed by refolding. Despite the lack of correct VLPs, the chimeric proteins induced high levels of antibodies in BALB/c mice. These antibodies specifically recognized either eukaryotically expressed hRBD or bacterially expressed N protein (2-220) of SARS-CoV-2. CTL-epitope-containing proteins were purified as VLPs. The production of cytokines was analyzed through flow cytometry after stimulation of T-cells with target CTL peptides. Only a protein with a deleted polyarginine (PA) domain was able to induce the specific activation of T-cells. At the same time, the T-cell response against the carrier HBc/G protein was detected for both proteins. The neutralization of SARS-CoV-2 pseudotyped murine retrovirus with anti-HBc/G-RBM sera was found to be low.

In situ bio-mineralized Mn nanoadjuvant enhances anti-influenza immunity of recombinant virus-like particle vaccines

Virus like particles (VLPs) have been well recognized as one of the most important vaccine platforms due to their structural similarity to natural viruses to induce effective humoral and cellular immune responses. Nevertheless, lack of viral nucleic acids in VLPs usually leads the vaccine candidates less efficient in provoking innate immune against viral infection. Here, we constructed a biomimetic dual antigen hybrid influenza nanovaccines THM-HA@Mn with robust immunogenicity via in situ synthesizing a stimulator of interferon genes (STING) agonist Mn3O4 inside the cavity of a recombinant Hepatitis B core antigen VLP (HBc VLP) having fused SpyTag and influenza M2e antigen peptides (Tag-HBc-M2e, THM for short), followed by conjugating a recombinant hemagglutinin (rHA) antigen on the surface of the nanoparticles through SpyTag/SpyCatcher ligating. Such inside Mn3O4 immunostimulator-outside rHA antigen design, together with the chimeric M2e antigen on the HBc skeleton, enabled the synthesized hybrid nanovaccines THM-HA@Mn to well imitate the spatial distribution of M2e/HA antigens and immunostimulant in natural influenza virus. In vitro cellular experiments indicated that compared with the THM-HA antigen without Mn3O4 and a mixture vaccine consisting of THM-HA+MnOx, the THM-HA@Mn hybrid nanovaccines showed the highest efficacies in dendritic cells uptake and in promoting BMDC maturation, as well as inducing expression of TNF-α and type I interferon IFN-β. The THM-HA@Mn also displayed the most sustained antigen release at the injection site, the highest efficacies in promoting the DC maturation in lymph nodes and germinal center B cells activation in the spleen of the immunized mice. The co-delivery of immunostimulant and antigens enabled the THM-HA@Mn nanovaccines to induce the highest systemic antigen-specific antibody responses and cellular immunogenicity in mice. Together with the excellent colloid dispersion stability, low cytotoxicity, as well as good biosafety, the synthetic hybrid nanovaccines presented in this study offers a promising strategy to design VLP-based vaccine with robust natural and adaptive immunogenicity against emerging viral pathogens.

Inactivated Poliovirus Vaccine: Recent Developments and the Tortuous Path to Global Acceptance.

Inactivated poliovirus vaccine (IPV), available since 1955, became the first vaccine to be used to protect against poliomyelitis. While the immunogenicity of IPV to prevent paralytic poliomyelitis continues to be irrefutable, its requirement for strong containment (due to large quantities of live virus used in the manufacturing process), perceived lack of ability to induce intestinal mucosal immunity, high cost and increased complexity to administer compared to oral polio vaccine (OPV), have limited its use in the global efforts to eradicate poliomyelitis. In order to harvest the full potential of IPV, a program of work has been carried out by the Global Polio Eradication Initiative (GPEI) over the past two decades that has focused on: (1) increasing the scientific knowledge base of IPV; (2) translating new insights and evidence into programmatic action; (3) expanding the IPV manufacturing infrastructure for global demand; and (4) continuing to pursue an ambitious research program to develop more immunogenic and safer-to-produce vaccines. While the knowledge base of IPV continues to expand, further research and product development are necessary to ensure that the program priorities are met (e.g., non-infectious production through virus-like particles, non-transmissible vaccine inducing humoral and intestinal mucosal immunity and new methods for house-to-house administration through micro-needle patches and jet injectors), the discussions have largely moved from whether to how to use this vaccine most effectively. In this review, we summarize recent developments on expanding the science base of IPV and provide insight into policy development and the expansion of IPV manufacturing and production, and finally we provide an update on the current priorities.

A J Paramyxovirus-vectored HIV vaccine induces humoral and cellular responses in mice

Human immunodeficiency virus (HIV) infects and depletes CD4+ T-cells, resulting in Acquired Immunodeficiency Syndrome (AIDS) and death. Despite numerous clinical trials, there is no licensed HIV vaccine. The HIV envelope glycoprotein (env) is a major target for vaccine development, especially for the development of antibody-mediated protection. In this study, we used J paramyxovirus (JPV) as a viral vector to express HIV-env. We replaced the JPV small hydrophobic (SH) gene with HIV-env (rJPV-env). Intranasal rJPV-env immunization induced anti-HIV-gp120 IgG antibodies in mice. Furthermore, we examined the immunogenicity of homologous and heterologous prime/boost regimens with rJPV-env, parainfluenza virus 5 (rPIV5)-vectored HIV-env, and HIV-Gag-Env virus-like particles (VLPs). The rJPV-env/rPIV5-env heterologous prime/boost regimen induced the strongest humoral and cellular responses. Introducing a third dose of immunization, mice that received a viral-vectored prime had high levels of HIV-env-specific cellular responses, with group rJPV-env/rPIV5-env/VLP having the highest. Together, this work indicates that a heterologous combination of viral-vectored HIV-env vaccines and a HIV-Gag-Env VLP induces high levels of humoral and cellular responses against HIV in mice.

Transmission and dynamics of mother-infant gut viruses during pregnancy and early life

Early development of the gut ecosystem is crucial for lifelong health. While infant gut bacterial communities have been studied extensively, the infant gut virome remains under-explored. To study the development of the infant gut virome over time and the factors that shape it, we longitudinally assess the composition of gut viruses and their bacterial hosts in 30 women during and after pregnancy and in their 32 infants during their first year of life. Using shotgun metagenomic sequencing applied to dsDNA extracted from Virus-Like Particles (VLPs) and bacteria, we generate 205 VLP metaviromes and 322 total metagenomes. With this data, we show that while the maternal gut virome composition remains stable during late pregnancy and after birth, the infant gut virome is dynamic in the first year of life. Notably, infant gut viromes contain a higher abundance of active temperate phages compared to maternal gut viromes, which decreases over the first year of life. Moreover, we show that the feeding mode and place of delivery influence the gut virome composition of infants. Lastly, we provide evidence of co-transmission of viral and bacterial strains from mothers to infants, demonstrating that infants acquire some of their virome from their mother’s gut.

Intranasal Ion-Triggered In Situ Delivery System of Virus-like Particles: Development Using the Quality by Design Approach.

The rapid growth in the prevalence of infectious diseases requires timely action from drug developers. In recent years, the COVID-19 pandemic has demonstrated the unpreparedness of the population for such emergencies. The introduction of modern methods of Design of Experiments (DoE) is required to accelerate the process of drug development and bring a drug to market. The main objective of this study was to develop an ion-triggered in situ system for intranasal delivery of VLP using a Quality by Design approach. Based on a literature review and initial studies, the key QTPP, CQA, CPP, and CMA were identified to develop a novel delivery system for virus-like particles. As a result of the studies on the quality attributes of the developed delivery system, an ion-triggered in situ gel meeting all the specified parameters was obtained using the Quality by Design method.

Serological characterisation of Lagovirus virus-like particles originating from native and mutated VP60 of rabbit haemorrhagic disease virus 2 and European brown hare syndrome virus.

Since lagoviruses cannot be cultivated in vitro, using expression systems is an alternative and promising way of producing diagnostic viral antigens. It opens up their use as active immunogens for vaccine production. Virus-like particles (VLPs) were produced in a baculovirus expression system in Spodoptera frugiperda 9 (Sf9) insect cells based on wild-type and mutated variants of the virus capsid VP60 protein from a Polish strain of European brown hare syndrome virus (EBHSV) and wild-type and mutated versions of this protein from a Polish strain of rabbit haemorrhagic disease virus 2 (RHDV2). The mutations were the substitution of an arginylglycylaspartic acid (Arg-Gly-Asp/RGD) motif in the P2 subdomain and, in the S or P2 domain, the substitution of three lysines. The VLPs were purified with sucrose gradient ultracentrifugation. Protein production was confirmed by Western blot analysis using rabbit or hare sera and ELISA tests with different types of monoclonal antibody. The haemagglutination properties of some VLPs were also evaluated. Electron microscopy of wild-type EBHSV, wild-type RHDV2 and the four VP60 variants produced in this experiment revealed the formation of characteristic VLP structures. For the first time, mutated VLPs of RHDV2 with an RGD motif in the VP60 sequence were obtained, which could potentially be used to deliver cargo to eukaryotic cells. Virus-like particles based on the VP60 proteins of EBHSV and RHDV with a three-lysine substitution in the S or P2 domains were also obtained. Potential exists for VLPs of EBHSV and RHDV2 as vaccine candidates.

Generation of porcine circovirus type 4 virus-like particles and their use to detect serum antibodies.

Porcine circovirus type 4 (PCV4), first identified in 2019 as a newly emerging pathogen, has been found in several provinces of China, as well as in Korea and Thailand. Since PCV4 is not included in immunization programs, epidemiological investigations should be conducted for detection of anti-PCV4 antibodies. Virus-like particles (VLPs) are frequently used for serological analysis of pathogen infections. However, there have been no reports on using PCV4 VLPs for serological investigation of PCV4 infection. In this study, we generated self-assembled PCV4 VLPs using an E. coli expression system, purified them using a two-step process, and used them to develop an indirect ELISA. This ELISA method was found to be highly specific, sensitive, and repeatable, making it suitable for PCV4 antibody detection in serum samples. Finally, the ELISA was used to analyze 422 serum samples collected from across several regions in China, 134 of which tested positive. Thus, the PCV4-VLP-based ELISA can effectively detect antibodies against PCV4 in serum samples, making it a useful tool for PCV4 epidemiology.