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Existing microbiota databases are biased toward adult samples, hampering accurate profiling of the infant gut microbiome. Here, we generated a metagenome-assembled genome inventory for children (MAGIC) from a large collection of bulk and viral-like particle-enriched metagenomes from 0 to 7 years of age, encompassing 3,299 prokaryotic and 139,624 viral species-level genomes, 8.5% and 63.9% of which are unique to MAGIC. MAGIC improves early-life microbiome profiling, with the greatest improvement in read mapping observed in Africans. We then identified 54 candidate keystone species, including several Bifidobacterium spp. and four phages, forming guilds that fluctuated in abundance with time. Their abundances were reduced in preterm infants and were associated with childhood allergies. By analyzing the B. longum pangenome, we found evidence of phage-mediated evolution and quorum sensing-related ecological adaptation. Together, the MAGIC database recovers genomes that enable characterization of the dynamics of early-life microbiomes, identification of candidate keystone species, and strain-level study of target species. [Display omitted] • MAGIC features 3,299 prokaryotic and 139,624 viral species-level genomes • MAGIC improves early-life microbiome profiling with extended genomic information • Candidate keystone species are associated with diseases, geography, and age • Extended Bifidobacterium longum pangenome reveals evolutionary insights Existing microbiota databases are biased toward adults, hindering infant gut microbiome profiling. Peng et al. introduce the metagenome-assembled genome inventory for children (MAGIC), featuring 3,299 prokaryotic and 139,624 viral genomes. MAGIC enables characterization of early-life microbiome dynamics, identification of candidate keystone species, and strain-level study of target species. [ABSTRACT FROM AUTHOR]

Introduction: The effects of viral infections might be apolipoprotein E (apoE) isoform-dependent. In humans, there are three major apoE isoforms, E2, E3, and E4. E4 is associated with the enhanced entry of several viruses into the brain and their disease progression. A concern of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the development of post-acute COVID-19 syndrome, also known as long COVID. Genetic risk factors for developing long COVID were reported. Methods: In this study, we used virus-like particles (VLPs) that include expression of the SARS-CoV-2 nucleocapsid (N), membrane (M), and envelope (E) structural proteins together with S. In the current study, we used human E2, E3, and E4 targeted replacement mice to assess whether these VLPs affect body weight, behavioral and cognitive performance, and circadian body temperatures. Using VLPs allow working outside an ABSL-3 facility. Results: The effects of VLPs on some behavioral measures were apoE isoform-dependent, with the E2 mice being more affected than E3 or E4 mice. The overall decreased activity in the open field containing objects in week 2 indicate that VLPs can also reduce activity levels in an apoE isoform-independent fashion. Discussion: The results of the current study indicate that even in the absence of viral replication, detrimental effects of VLPs on behavioral measures and circadian body temperatures are seen. [ABSTRACT FROM AUTHOR]

Among vaccine platforms against SARS-CoV-2, the virus-like particles (VLP) hold advantageous immunological and safety attributes. Furthermore, there are barriers to the simultaneous and rapid monitoring of critical parameters throughout associated processes to ensure biodrug quality. This work is pioneering in advanced monitoring for producing SARS-CoV-2 VLP by the baculovirus/Sf9 insect cell system, using mid-infrared spectroscopy. It aimed to establish chemometric models based on spectra in offline attenuated total reflectance (ATR) and transmittance capture modes using partial least squares (PLS) and artificial neural network (ANN) for the biochemical monitoring (Viable cell density (X V), cell viability; glucose, glutamine, glutamate, lactate, NH 4 + concentrations, and virus titer) of SARS-CoV-2 VLP production. Monocistronic recombinant baculovirus carrying genes of SARS-CoV-2 structural proteins and receptor binding domain spike protein (RBD) were used for VLP production by single or combined infections. Among assessed spectral segments, the complete spectral range was the most suitable. The results obtained by both models were mostly similar. However, ANN-created models stood out, providing the best performances, especially when the ATR capture mode was used. This becomes particularly evident in the X V , viral titer, and glucose parameters, which showed statistically significant differences, and lower relative error values of 27,5; 5,4, and 2,4 %, respectively. [Display omitted] • Attenuated total reflection mid-infrared is useful for Sf9 cell bioprocess tracking. • Spectra filtering choice showed relevance in models' precision for monitoring. • Linear and non-linear regression models performed similarly for most biochemicals. • Neural network regression was better suited for cell density, viral titer, and glucose. • Virus titer Log 10 transformation improved markedly the modeling prediction quality. [ABSTRACT FROM AUTHOR]

Background: Allergen‐carrying virus‐like particles are effective and safe means of allergen immunotherapy (AIT) in rodent models. Objective: To study the development of allergen‐blocking immunoglobulin (Ig)G in dogs injected with Der f 2‐carrying enveloped plant‐based bioparticles (eBPs). Materials and Methods: Laboratory beagle dogs were injected intradermally (ID) or subcutaneously (SC) with Der f 2‐eBP three times at 2‐week intervals. A basophil mediator release assay was used to compare the reactivity of Der f 2‐eBPs to that of recombinant Der f 2. Allergen‐specific IgG serum levels were determined by immunoblotting and ELISA. The allergen‐blocking potential of postvaccination IgG was assessed by Pet Allergy Xplorer (PAX) macroarray and basophil mediator release inhibition assays. Results: The amount of Der f 2 eBPs needed to induce basophil activation was 1000‐fold higher than that of the soluble natural allergen. In both immunisation groups, eBP injections caused no adverse events and induced Der f 2‐specific IgG, first detected on Day (D)14 and peaking on D41. The co‐incubation of sera with a Der f 2‐IgE‐rich canine serum pool resulted in a mean PAX inhibition of 70% (ID) to 80% (SC) on D41. For both groups, the inhibition of basophil mediator release reached 75% on D28 and D41. The percentage inhibition of PAX and mediator release correlated significantly with Der f 2 IgG levels. Conclusion and Clinical Relevance: Intradermal and subcutaneous injections of Der f 2‐eBPs were safe and increased Der f 2‐specific IgG. The clinical benefit of immunotherapy will be evaluated in future trials enrolling atopic dogs allergic to house dust mites. [ABSTRACT FROM AUTHOR]

Background: The garden dormouse (Eliomys quercinus) has experienced a significant population decline across Europe in recent decades. While habitat loss and climate change are often cited as primary factors, pathogen exposure, either to novel or to previously known, may play a role in such a decline. This study aimed to investigate the presence of polyomaviruses in garden dormice, given that these viruses are highly prevalent and can cause disease, particularly in immunocompromised individuals. Methods: The carcasses of garden dormice (n = 89) were collected throughout Germany. Kidney samples were tested for the presence of polyomavirus DNA using nested degenerate and specific diagnostic PCRs. Seroprevalence was assessed from chest cavity fluid samples through an enzyme-linked immunosorbent assay using polyomavirus VP1 virus-like particles produced in yeast. Results: A new polyomavirus, related to chimpanzee (Pan troglodytes) polyomaviruses 4 and 5 and human Merkel cell polyomavirus, was identified in the garden dormouse. Two 5,380 bp-length complete viral genomes were sequenced from dormice kidney samples (sequences PQ246041 and PQ246042). Genes encoding the putative structural proteins VP1, VP2, and VP3, as well as the Large, Middle, and small T antigens, containing conserved functional domains were identified. Polyomavirus DNA was detected in 2 of 74 dormice (2.7%, 95% confidence interval: 0-6.4%) through PCR, while 12 of 69 animals (17.4%, 95% confidence interval: 8.4-26.3%) tested positive for polyomavirus-specific antibodies. Conclusions: In conclusion, here we describe a novel polyomavirus in the garden dormouse with molecular and serological detection. Pairwise sequence comparison and phylogenetic analysis suggest that this novel virus may represent a novel species within the genus Alphapolyomavirus. Future work should examine if this virus is garden dormouse-specific and whether it is associated with disease in dormice. [ABSTRACT FROM AUTHOR]

Human norovirus (HuNoV) accounts for over 700 million cases of gastroenteritis annually. Episodes of HuNoV disease are characterized by vomiting and diarrhea as the two most prominent symptoms. Despite its prevalence, our understanding of the pathophysiological mechanisms triggered upon HuNoV infection is limited, mainly due to a lack of suitable animal models. Our aim was to use the recent HuNoV zebrafish larvae model to study the effect of HuNoV infection on intestinal motility and investigate whether one viral protein could act as an enterotoxin, as seen with rotavirus. We studied whether HuNoV infection affects the contraction frequency of the intestinal bulb and the posterior intestine as well as the transit time. Infection of larvae, following injection of a HuNoV GII.4-containing stool sample in the yolk, resulted in an increased contraction frequency in the intestinal bulb. A comparable effect was observed in serotonin-treated larvae, corresponding to the natural function of serotonin. The higher replication efficacy of HuNoV GII.4 likely explains why they have a more marked effect on gut motility, when compared to other genotypes. Additionally, transit time of fluorescent food was prolonged in HuNoV GII.4 infected larvae, suggesting a loss of coordination in bowel movements upon infection. To identify the proteins responsible for the effect, individual HuNoV non-structural proteins and virus-like particles (VLPs) were injected intraperitoneally (ip). VLPs carrying VP1/VP2, but not those with only VP1, induced increased contraction frequencies in the intestinal bulb in a dose-dependent manner. In conclusion, our findings suggest that the viral capsid and potentially the minor capsid protein VP2 play a crucial role in the aetiology of symptoms associated with HuNoV, potentially acting as a viral enterotoxin. This work contributes to the understanding of the pathophysiological mechanisms in HuNoV-induced disease and further attests zebrafish as a valuable HuNoV disease model. Author summary: Human norovirus causes more than 700 million cases of gastroenteritis every year. Episodes of HuNoV disease are characterized by vomiting and diarrhea as the two most prominent symptoms. Despite its global prevalence, our understanding of the underlying mechanisms causing human norovirus disease are limited due to a historical lack of proper animal models. Recently, we developed a new small animal model for human norovirus in zebrafish larvae. Here, we used the optical transparency during the early larval stages of zebrafish larvae to study the effect of human norovirus infection on the intestinal motility and transit time, and to potentially identify a new viral enterotoxin, as has been done for human rotavirus. We found that the contraction frequency of a specific part of the zebrafish intestine, the intestinal bulb, increased upon human norovirus infection. Additionally, human norovirus infection caused a prolonged transit time after feeding, indicating a loss of coordination in bowel movements upon infection. Finally, we show that it are the viral capsid proteins that play a crucial role in inducing these effects. Overall, our study provides new insights into the disease mechanism of human norovirus. [ABSTRACT FROM AUTHOR]

The Baculovirus Expression Vector System (BEVS) is highly valued in vaccine development, protein engineering, and drug metabolism research due to its biosafety, operational convenience, rapid scalability, and capacity for self-assembling virus-like particles. However, increasing cell density at the time of inoculation severely compromises the production capacity of BEVS, resulting in the "cell density effect". This study aimed to explore the mechanisms of the cell density effect through time-series analysis of transcriptomes and proteomes, with the goal of overcoming or alleviating the decline in productivity caused by increased cell density. The dynamic analysis of the omics of High Five cells under different CCI (cell density at infection) conditions showed that the impact of the "cell density effect" increased over time, particularly affecting genetic information processing, error repair, protein expression regulation, and material energy metabolism. Omics analysis of the growth stage of High Five cells showed that after 36 h of culture (cell density of about 1 × 106 cells/mL), the expression of ribosome-related proteins decreased, resulting in a rapid decrease in protein synthesis capacity, which was a key indicator of cell aging. Senescence verification experiments showed that cells began to show obvious early aging characteristics after 36 h, resulting in a decrease in the host cell's ability to resist stress. Overexpression and siRNA inhibition studies showed that the ndufa12 gene was a potential regulatory target for restricting the "cell density effect". Our results suggested that stress-induced premature senescence in High Five cell cultures, resulting in reduced energy metabolism and protein synthesis capabilities, was a critical factor contributing to cell density effects, and ultimately affecting virus production. In conclusion, this study provided new insights into managing virus production limitations due to cell density effects and offered innovative strategies to mitigate the adverse effects of cellular aging in biomanufacturing technologies. [ABSTRACT FROM AUTHOR]

All lineages of SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, contain mutations between amino acids 199 and 205 in the nucleocapsid (N) protein that are associated with increased infectivity. The effects of these mutations have been difficult to determine because N protein contributes to both viral replication and viral particle assembly during infection. Here, we used single-cycle infection and virus-like particle assays to show that N protein phosphorylation has opposing effects on viral assembly and genome replication. Ancestral SARS-CoV-2 N protein is densely phosphorylated, leading to higher levels of genome replication but 10-fold lower particle assembly compared to evolved variants with low N protein phosphorylation, such as Delta (N:R203M), Iota (N:S202R), and B.1.2 (N:P199L). A new open reading frame encoding a truncated N protein called N*, which occurs in the B.1.1 lineage and subsequent lineages of the Alpha, Gamma, and Omicron variants, supports high levels of both assembly and replication. Our findings help explain the enhanced fitness of viral variants of concern and a potential avenue for continued viral selection. Author summary: The COVID-19 pandemic was caused by SARS-CoV-2 and is characterized by waves of evolved viral variants with mutations across the viral genome. Nucleocapsid (N) is a viral protein that is critical for viral RNA packaging into particles and for viral replication in cells. Over the pandemic, N has acquired several mutations between amino acids 199 and 205 that are associated with increased infectivity. The mechanism underlying these mutations has been difficult to elucidate given the multiple roles of N protein in the viral lifecycle. Here, we used developed methods to separately determine the effect of mutations on viral particle assembly vs viral RNA replication. We found that the mutations between amino acids 199 and 205 affect N protein phosphorylation and have opposite effects on viral assembly and genome replication. Ancestral SARS-CoV-2 N protein is densely phosphorylated, leading to higher levels of genome replication but 10-fold lower particle assembly compared to evolved variants with low N protein phosphorylation. One of the mutations found in the Alpha, Gamma, and Omicron variants of concern results in expression of a new, truncated N protein called N* that supports high levels of both assembly and replication. Our findings help explain the enhanced fitness of viral variants of concern and a potential avenue for continued viral selection. [ABSTRACT FROM AUTHOR]

RTS,S and R21 are the only vaccines recommended by the WHO to protect children from Plasmodium falciparum (Pf) clinical malaria. Both vaccines target the Pf sporozoite surface protein circumsporozoite protein (CSP). Recent studies showed that human antibodies neutralize Pf sporozoites most efficiently when simultaneously binding to the PfCSP NANP repeat and the NPDP junction domain. However, neither RTS,S nor R21 targets this junction domain. To test the potential of the NPDP junction domain and other sites of PfCSP as innovative vaccine targets, we developed multiple vaccine candidates based on cucumber mosaic virus-like particles (CuMVTT-VLPs). These candidates vary in several aspects: the number of targeted NANP repeats, the presence or absence of the junction domain, the cleavage site, and up to three NVDP repeats within the target sequence. Immunogenicity and efficacy studies were conducted in BALB/c mice, utilizing chimeric Plasmodium berghei (Pb) sporozoites, in which the endogenous CSP has been replaced by PfCSP (Pb/PfCSP). We observed a positive association between the number of targeted NANP repeats and the induction of specific IgM/IgG antibodies. Elevated humoral responses led to enhanced protection against parasitemia after Pb/PfCSP sporozoite challenge. Especially high-avidity/affinity antibody formation and vaccine protection were NANP repeat-dependent. Intriguingly, vaccine efficacy was not enhanced by targeting sites on PfCSP other than the NANP repeats. Our data emphasize the dominant role of the NANP repeat region for induction of protective antibodies. Furthermore, we present here novel malaria vaccine candidates with an excellent immunogenic profile that confer sterile protection in mice, even in absence of adjuvants. [ABSTRACT FROM AUTHOR]

This review addresses the ongoing global challenge posed by emerging and evolving viral diseases, underscoring the need for innovative vaccine development strategies. It focuses on the modern approaches to creating vaccines based on recombinant proteins produced in different expression systems, including bacteria, yeast, plants, insects, and mammals. This review analyses the advantages, limitations, and applications of these expression systems for producing vaccine antigens, as well as strategies for designing safer, more effective, and potentially 'universal' antigens. The review discusses the development of vaccines for a range of viral diseases, excluding SARS-CoV-2, which has already been extensively studied. The authors present these findings with the aim of contributing to ongoing research and advancing the development of antiviral vaccines. [ABSTRACT FROM AUTHOR]

[Display omitted] • Modular bioengineered nanofibrils neutralize SARS-CoV-2 virus-like particles. • Recombinant protein subunits spontaneously assemble into functional fibrils. • The assembled amyloid-like nanofibrils are biocompatible. • Mesoscopic structure and activity of the protein assembly are easily modulated. • Surfaces coated with nanofibrils capture SARS-CoV-2 Spike in wet environments. The recent coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spurred intense research efforts to develop new materials with antiviral activity. In this study, we genetically engineered amyloid-based nanofibrils for capturing and neutralizing SARS-CoV-2. Building upon the amyloid properties of a short Sup35 yeast prion sequence, we fused it to SARS-CoV-2 receptor-binding domain (RBD) capturing proteins, LCB1 and LCB3. By tuning the reaction conditions, we achieved the spontaneous self-assembly of the Sup35-LCB1 fusion protein into a highly homogeneous and well-dispersed amyloid-like fibrillar material. These nanofibrils exhibited high affinity for the SARS-CoV-2 RBD, effectively inhibiting its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor, the primary entry point for the virus into host cells. We further demonstrate that this functional nanomaterial entraps and neutralizes SARS-CoV-2 virus-like particles (VLPs), with a potency comparable to that of therapeutic antibodies. As a proof of concept, we successfully fabricated patterned surfaces that selectively capture SARS-CoV-2 RBD protein on wet environments. Collectively, these findings suggest that these protein-only nanofibrils hold promise as disinfecting coatings endowed with selective SARS-CoV-2 neutralizing properties to combat viral spread or in the development of sensitive viral sampling and diagnostic tools. [ABSTRACT FROM AUTHOR]

[Display omitted] Remarkable progress has been made in tumour immunotherapy in recent decades. However, the clinical outcomes of therapeutic interventions remain unpredictable, largely because of inefficient immune responses. To address this challenge and optimise immune stimulation, we present a novel administration route for enhancing the bioavailability of immunotherapeutic drugs. Our approach involves the development of an oral tumour vaccine utilising virus-like particles derived from the Hepatitis B virus core (HBc) antigen. The external surfaces of these particles are engineered to display the model tumour antigen OVA, whereas the interiors are loaded with cytosine phosphoguanosine oligodeoxynucleotide (CpG ODN), resulting in a construct called CpG@OVAHBc with enhanced antigenicity and immune response. For oral delivery, CpG@OVAHBc is encapsulated in a crosslinked dextran hydrogel called CpG@OVAHBc@Dex. The external hydrogel shield safeguards the biomimetic virus particles from degradation by gastric acid and proteases. Upon exposure to intestinal flora, the hydrogel disintegrates, releasing CpG@OVAHBc at the intestinal mucosal site. Owing to its virus-like structure, CpG@OVAHBc exhibits enhanced adhesion to the mucosal surface, facilitating uptake by microfold cells (M cells) and subsequent transmission to antigen-presenting cells. The enzyme-triggered release of this oral hydrogel ensures the integrity of the tumour vaccine within the digestive tract, allowing targeted release and significantly improving bioavailability. Beyond its efficacy, this oral hydrogel vaccine streamlines drug administration, alleviates patient discomfort, and enhances treatment compliance without the need for specialised injection methods. Consequently, our approach expands the horizons of vaccine development in the field of oral drug administration. [ABSTRACT FROM AUTHOR]

Injectable hydrogels represent a promising strategy for the extended release of biological molecules, thereby reducing the frequency of injections. This study introduces a novel system based on Michael addition of dextran and polyethylene glycol (PEG) polymers functionalized with oxanorbornadiene (OND) and thiol groups, respectively. Reliable control over gelation speed allows administration by injection using a simple syringe‐to‐syringe mixing protocol that entrains more than 95% of virus‐like particle (VLP) cargo. A combination of retro‐Diels‐Alder and hydrolytic ester bond cleavage gives rise to programmable release of the VLPs. Different release profiles, including burst, linear, and delayed release over a two‐week period, are engineered using different OND linkages, and rheological characterization shows the hydrogels to be well within the desired range of stiffness for subcutaneous use. The modular nature of this system offers a generalizable platform for developing degradable materials aimed at sustained release biomedical applications. [ABSTRACT FROM AUTHOR]

Classically, all hepatitis E virus (HEV) variants causing human infection belong to the genus Paslahepevirus (HEV-A). However, the increasing cases of rat HEV infection in humans since 2018 challenged this dogma, posing increasing health threats. Herein, we investigated the underlying mechanisms dictating the zoonotic potentials of different HEV species and their possible cross-protection relationships. We found that rat HEV virus-like particles (HEVVLPs) bound to human liver and intestinal cells/tissues with high efficiency. Moreover, rat HEVVLPs and infectious rat HEV particles penetrated the cell membrane and entered human target cells postbinding. In contrast, ferret HEVVLPs showed marginal cell binding and entry ability, bat HEVVLPs and avian HEVVLPs exhibited no binding and entry potency. Structure-based three-dimensional mapping identified that the surface spike domain of rat HEV is crucial for cell binding. Antigenic cartography indicated that rat HEV exhibited partial cross-reaction with HEV-A. Intriguingly, sera of HEV-A infected patients or human HEV vaccine Hecolin® immunized individuals provided partial cross-protection against the binding of rat HEVVLPs to human target cells. In summary, the interactions between the viral capsid and cellular receptor(s) regulate the distinct zoonotic potentials of different HEV species. The systematic characterization of antigenic cartography and serological cross-reactivity of different HEV species provide valuable insights for the development of species-specific diagnosis and protective vaccines against zoonotic HEV infection. [ABSTRACT FROM AUTHOR]

An effective vaccine against hepatitis C virus (HCV) should elicit both humoral and cellular immune responses. Previously, we characterized a bivalent vaccine candidate against hepatitis B (HBV) and HCV using chimeric HBV-HCV virus-like particles (VLP), in which the highly conserved epitope of HCV E2 glycoprotein (residues 412–425) was inserted into the hydrophilic loop of HBV small surface antigen (sHBsAg). While sHBsAg_412–425 elicited cross-neutralizing antibodies, it did not trigger a T-cell response against HCV. Thus, this study aimed to develop a vaccine candidate engaging both arms of adaptive immune response, potentially offering stronger protection against HCV. We evaluated the immunogenicity of minicircle (MC) DNA vaccines encoding sHBsAg_412–425 and HCV nonstructural (NS) proteins in BALB/c mice. Co-administration of sHBsAg_412–425 and NS induced a potent T-cell response, especially against NS3 and high titers of antibodies specific to HCV E2. Additionally, these antibodies recognized native HCV envelope glycoprotein heterodimers (E1E2) across multiple HCV genotypes and showed binding profiles to E1E2 alanine mutants comparable to the broadly neutralizing AP33 antibody. Overall, the findings demonstrate that MC DNA vaccine incorporating both sHBsAg_412–425 and HCV NS protein sequences induces robust, T-cell and AP33-like antibody responses, highlighting its potential as pan-genotypic prophylactic vaccine against HCV. [ABSTRACT FROM AUTHOR]

Soybean bradyrhizobia (Bradyrhizobium spp.) are symbiotic root-nodulating bacteria that fix atmospheric nitrogen for the host plant. The University of Delaware Bradyrhizobium Culture Collection (UDBCC; 353 accessions) was created to study the diversity and ecology of soybean bradyrhizobia. Some UDBCC accessions produce temperate (lysogenic) bacteriophages spontaneously under routine culture conditions without chemical or other apparent inducing agents. Spontaneous phage production may promote horizontal gene transfer and shape bacterial genomes and associated phenotypes. A diverse subset (n = 98) of the UDBCC was examined for spontaneously produced virus-like particles (VLPs) using epifluorescent microscopy, with a majority (69%) producing detectable VLPs (>1 × 107 mL−1) in laboratory culture. Phages from the higher-producing accessions (>2.0 × 108 VLP mL−1; n = 44) were examined using transmission electron microscopy. Diverse morphologies were observed, including various tail types and lengths, capsid sizes and shapes, and the presence of collars or baseplates. In many instances, putative extracellular vesicles of a size similar to virions were also observed. Three of the four species examined (B. japonicum, B. elkanii, and B. diazoefficiens) produced apparently tailless phages. All species except B. ottawaense also produced siphovirus-like phages, while all but B. diazoefficiens additionally produced podovirus-like phages. Myovirus-like phages were restricted to B. japonicum and B. elkanii. At least three strains were polylysogens, producing up to three distinct morphotypes. These observations suggest spontaneously produced phages may play a significant role in the ecology and evolution of soybean bradyrhizobia. [ABSTRACT FROM AUTHOR]

Oncolytic measles virus (MeV) is a promising anti-cancer treatment. However, the production of high titers of infectious MeV (typically 107–109 TCID50 per dose) is challenging because the virus is unstable under typical production conditions. The objective of this study was to investigate how the multiplicity of infection (MOI) and different media—a serum-containing medium (SCM), a serum-free medium (SFM) and two chemically defined media (CDM)—affect MeV production. We infected Vero cells at MOIs of 0.02, 0.2 or 2 TCID50 cell−1 and the lowest MOI resulted in the largest number of infected cells towards the end of the production period. However, this did not equate to higher maximum MeV titers, which were similar for all the MOIs. The medium had a moderate effect, generating maximum titers of 0.89–2.17 × 106, 1.08–1.25 × 106 and 4.58–9.90 × 105 TCID50 mL−1 for the SCM, SFM and CDM, respectively. Infection at a low MOI often required longer process times to reach maximum yields. On the other hand, a high MOI requires a large amount of MeV stock. We would therefore recommend a mid-range MOI of 0.2 TCID50 cell−1 for MeV production. Our findings show that SCM, SFM and CDM are equally suitable for MeV production in terms of yield and process time. This will allow MeV production in serum-free conditions, addressing the safety risks and ethical concerns associated with the use of serum. [ABSTRACT FROM AUTHOR]

Glycoconjugate vaccines have been effective in preventing numerous bacterial infectious diseases and have shown recent potential to treat cancers through active immunotherapy. Soluble polysaccharides elicit short-lasting immune responses and are usually covalently linked to immunogenic carrier proteins to enhance the antigen-specific immune response by stimulating T-cell-dependent mechanisms. Nonetheless, the conjugation of purified polysaccharides to carrier proteins complexifies vaccine production, and immunization with protein glycoconjugates can lead to the undesirable immunogenic interference of the carrier. Recently, the use of nanoparticles and nanoassemblies for the delivery of antigenic saccharides has gathered attention from the scientific community. Nanoparticles can be easily functionalized with a diversity of functionalities, including T-cell epitope, immunomodulator and synthetic saccharides, allowing for the modulation and polarization of the glycoantigen-specific immune response. Notably, the conjugation of glycan to nanoparticles protects the antigens from degradation and enhances their uptake by immune cells. Different types of nanoparticles, such as liposomes assembled from lipids, inorganic nanoparticles, virus-like particles and dendrimers, have been explored for glycovaccine design. The versatility of nanoparticles and their ability to induce robust immune responses make them attractive delivery platforms for antigenic saccharides. The present review aims at summarizing recent advancements in the use of nano-scaled systems for the delivery of synthetic glycoantigens. After briefly presenting the immunological mechanisms required to promote a robust immune response against antigenic saccharides, this review will offer an overview of the current trends in the nanoparticle-based delivery of glycoantigens. [ABSTRACT FROM AUTHOR]

The development of effective vaccines necessitates a delicate balance between maximizing immunogenicity and minimizing safety concerns. Subunit vaccines, while generally considered safe, often fail to elicit robust and durable immune responses. Nanotechnology presents a promising approach to address this dilemma, enabling subunit antigens to mimic critical aspects of native pathogens, such as nanoscale dimensions, geometry, and highly repetitive antigen display. Various expression systems, including Escherichia coli (E. coli), yeast, baculovirus/insect cells, and Chinese hamster ovary (CHO) cells, have been explored for the production of nanoparticle vaccines. Among these, E. coli stands out due to its cost-effectiveness, scalability, rapid production cycle, and high yields. However, the E. coli manufacturing platform faces challenges related to its unfavorable redox environment for disulfide bond formation, lack of post-translational modifications, and difficulties in achieving proper protein folding. This review focuses on molecular and protein engineering strategies to enhance protein solubility in E. coli and facilitate the in vitro reassembly of virus-like particles (VLPs). We also discuss approaches for antigen display on nanocarrier surfaces and methods to stabilize these carriers. These bioengineering approaches, in combination with advanced nanocarrier design, hold significant potential for developing highly effective and affordable E. coli-derived nanovaccines, paving the way for improved protection against a wide range of infectious diseases. [ABSTRACT FROM AUTHOR]

Background: Cervical cancer ranks as the fourth most common cancer affecting women globally, with HPV as the primary etiology agent. Prophylactic HPV vaccines have substantially reduced the incidence of cervical cancer. Methods: This study assessed the immunogenicity of SCT1000, a 14-valent recombinant virus-like particle (VLP) vaccine developed by Sinocelltech, Ltd. using pseudovirion-based neutralization assays (PBNAs) and total IgG Luminex immunoassays (LIAs). Currently in phase III clinical trials in China, SCT1000 targets the same HPV types as Gardasil 9®, plus five additional high-risk types, thereby covering twelve high-risk HPV types implicated in 96.4% of cervical cancer cases. Results: In murine models, a dose of 1.85 μg per mouse was identified as optimal for evaluating SCT1000's immunogenicity in a three-dose regimen, as measured by PBNA and total IgG LIA across all 14 HPV types. SCT1000 induced high levels of protective antibodies, which were sustained for at least four months following the third dose. The vaccine also demonstrated stable and consistent immunogenicity in mouse potency assays under both long-term and accelerated conditions. Additionally, our studies revealed a strong correlation between the two serological tests used. Conclusions: SCT1000 elicited robust, durable, and consistent humoral immune responses across all 14 HPV types, indicating its potential as a broad-spectrum vaccine candidate against HPV types 6/11/16/18/31/33/35/39/45/51/52/56/58/59. The significant correlations observed between PBNA and total IgG LIA support the use of the Luminex-based total IgG method as a reliable and effective alternative for immunogenicity assessment in preclinical and future clinical vaccine development. [ABSTRACT FROM AUTHOR]

Moschus chrysogaster viral hemorrhagic disease (McVHD), caused by the Moschus chrysogaster hemorrhagic disease virus (McHDV), is an acute and highly fatal infectious disease of musk deer. At present, there is no prevention or treatment for this disease. In this study, we constructed a recombinant bacmid containing the McHDV VP60 gene and obtained the recombinant baculovirus rBac-McHDV VP60 by transfection into Sf9 (Spodoptera frugiperda) insect cells. The McHDV VP60 protein was successfully expressed in the insect cell-baculovirus expression system; furthermore, it was released in the supernatant of infected insect cells and spontaneously assembled to form virus-like particles (VLPs), which were structurally and immunologically indistinguishable from the Moschus chrysogaster viral hemorrhagic disease virion. Hypodermic vaccination of rabbits with the VLPs conferred complete protection in 14 days; this protection was found to be effective from the seventh day after VLP injection and was accompanied by a strong humoral response. This study is the first attempt to express the VP60 gene of McHDV using an insect baculovirus system, which provides an experimental basis for the virus-like particle vaccine of McVHD. [ABSTRACT FROM AUTHOR]

Currently available seasonal influenza vaccines confer variable protection due to antigenic changes resulting from the accumulation of diverse mutations. The analysis of new seasonal influenza vaccines is challenging in part due to the limitations of the traditional hemagglutination inhibition (HAI) assay with A/H3N2 strains. An improved and objective novel HAI assay was developed with recombinant virus-like particles (VLPs) and an egg-derived virus as agglutinins, the oseltamivir treatment of VLPs, human red blood cells, and using an automated image reader-based analysis of hemagglutination. HAI validation was demonstrated using four VLPs and egg-derived strains, with 46–56 serum samples tested 12 times in duplicate per strain. The validated HAI assay was precise as indicated by the percent geometric coefficient of variation for intra-, inter-, and total assay precision, as well as accurate as evidenced by percent bias measurements. The assay exhibited linearity, specificity for homologous type/subtype strains, and sensitivity with a starting dilution of 1:10. Assay robustness and sample stability were demonstrated as a percentage difference compared to reference condition. Validated HAI results were equivalent for the single and duplicate sample testing and correlated well with a qualified live wild-type influenza microneutralization assay. These findings demonstrate the suitability of this high-throughput novel modified validated HAI assay for evaluating vaccine immunogenicity and efficacy. [ABSTRACT FROM AUTHOR]

Goose-derived Newcastle disease (ND) and gosling plague (GP) are serious threats to the goose industry. Conventional vaccines have made significant contributions to preventing GP and ND. Nevertheless, the renewal of conventional vaccines and the application of novel vaccines are urgently needed to align with eco-friendly and efficient breeding concepts and achieve the final goal of epidemic purification. Therefore, based on the Newcastle disease virus-like particles (ND VLPs) vector platform, we developed novel chimeric ND-GP bivalent cVLPs (ND-GP cVLPs) displaying the NDV HN protein and the GPV VP3 protein. In vivo, immunization experiments revealed that geese immunized with 30 µg, 50 µg, or 70 µg of the ND-GP cVLPs and commercial vaccines produced highly effective hemagglutination inhibitory antibodies against NDV and neutralizing antibodies against GPV, respectively. Furthermore, 70 µg of the ND-GP cVLPs effectively protected against virulent NDV and GPV, reducing tissue damage from viral infection and virus shedding in the oropharynx and cloaca. In conclusion, we provide eco-friendly and efficient novel ND-GP cVLPs for preventing goose-derived ND and GP. Our findings provide the basis for using ND VLPs as foreign protein carriers for the developing of multi-conjugate vaccines. [ABSTRACT FROM AUTHOR]

Bats are tolerant to highly pathogenic viruses such as Marburg, Ebola, and Nipah, suggesting the presence of a unique immune tolerance toward viral infection. Here, we compared severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of human and bat (Rhinolophus ferrumequinum) pluripotent cells and fibroblasts. Since bat cells do not express an angiotensin-converting enzyme 2 (ACE2) receptor that allows virus infection, we transduced the human ACE2 (hA) receptor into the cells and found that transduced cells can be infected with SARS-CoV-2. Compared to human embryonic stem cells-hA, infected bat induced Pluripotent Stem Cells (iPSCs)-hA produced about a 100-fold lower level of infectious virus and displayed lower toxicity. In contrast, bat embryonic fibroblast-hA produced no infectious virus while being infectable and synthesizing viral RNA and proteins, suggesting abortive infection. Indeed, electron microscopy failed to detect virus-like particles in infected bat fibroblasts in contrast to bat iPSCs or human cells, consistent with the latter producing infectious viruses. This suggests that bat somatic but not pluripotent cells have an effective mechanism to control virus replication. Consistent with previous results by others, we find that bat cells have a constitutively activated innate immune system, which might limit SARS-CoV-2 infection compared to human cells. [ABSTRACT FROM AUTHOR]

Vaccination has played a critical role in mitigating COVID-19. Despite the availability of licensed vaccines, there remains a pressing need for improved vaccine platforms that provide high protection, safety, and versatility, while also reducing vaccine costs. In response to these challenges, our aim is to create a self-adjuvanted vaccine against SARS-CoV-2, utilizing Virus-Like Particles (VLPs) as the foundation. To achieve this, we produced bacteriophage (Qβ) VLPs in a prokaryotic system and purified them using a rapid and cost-effective strategy involving organic solvents. This method aims to solubilize lipids and components of the cell membrane to eliminate endotoxins present in bacterial samples. For vaccine formulation, Receptor Binding Domain (RBD) antigens were conjugated using chemical crosslinkers, a process compatible with Good Manufacturing Practice (GMP) standards. Transmission Electron Microscopy (TEM) confirmed the expected folding and spatial configuration of the QβVLPs vaccine. Additionally, vaccine formulation assessment involved SDS-PAGE stained with Coomassie Brilliant Blue, Western blotting, and stereomicroscopic experiments. In vitro and in vivo evaluations of the vaccine formulation were conducted to assess its capacity to induce a protective immune response without causing side effects. Vaccine doses of 20 µg and 50 µg stimulated the production of neutralizing antibodies. In in vivo testing, the group of animals vaccinated with 50 µg of vaccine formulation provided complete protection against virus infection, maintaining stable body weight without showing signs of disease. In conclusion, the QβVLPs-RBD vaccine has proven to be effective and safe, eliminating the necessity for supplementary adjuvants and offering a financially feasible approach. Moreover, this vaccine platform demonstrates flexibility in targeting Variants of Concern (VOCs) via established conjugation protocols with VLPs. [ABSTRACT FROM AUTHOR]

Background Rapidly evolving RNA viruses, such as human norovirus, generate extraordinary sequence diversity, posing a significant challenge to vaccine design. This diversity, coupled with short-lasting natural immunity, leads to reinfection throughout one's lifetime. How reexposure shapes humoral immunity to future norovirus strains remains incompletely understood. Methods We profiled the antibody responses following 2 community gastroenteritis outbreaks with GII.2 and GII.6 noroviruses in 1971. Using diverse virus-like particles (VLPs), enzyme-linked immunosorbent assay (ELISA), and carbohydrate-blocking assays (surrogate for neutralization), we examined the antibody response at acute and convalescent timepoints following GII.6 infection. Results Convalescent sera displayed strong homologous blocking, demonstrating a 5-fold increase in GII.6 carbohydrate blockade over acute samples, and broad blocking of diverse archival and modern GII.6 noroviruses. Convalescent sera displayed limited carbohydrate blocking of heterotypic VLPs, despite high ELISA binding titers. Select individuals developed broad cross-genotype blockade, but this response was established before the second outbreak. Finally, we applied a novel competitive carbohydrate-blocking assay to demonstrate the epitope specificity and discrete compartments of the neutralizing response. Conclusions Our data show that infection generates narrow, focused immunity directed toward the infecting genotype. We did detect broad cross-blocking in specific individuals, but these responses could be attributed to diverse, genotype-specific antibodies predating GII.6 infection. [ABSTRACT FROM AUTHOR]

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 wholegenome 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. [ABSTRACT FROM AUTHOR]

The retina-specific ABCA transporter, ABCA4, is essential for vision, and its genetic variants are associated with a wide range of inherited retinal degenerative diseases, leading to blindness. Of the 1630 identified missense variants in ABCA4, -50% are of unknown pathogenicity (variants of unknown significance, VUS). This genetic uncertainty presents three main challenges: (i) inability to predict disease-causing variants in relatives of inherited retinal degenerative disease patients with multiple ABCA4 mutations; (ii) limitations in developing variant-specific treatments; and (iii) difficulty in using these variants for future disease prediction, affecting patients' lifeplanning and clinical trial participation. To unravel the clinical significance of ABCA4 genetic variants at the level of protein function, we have developed a virus-like particle-based system that expresses the ABCA4 protein and its variants. We validated the efficacy of this system in the enzymatic characterization (ATPase activity) of VLPs harboring ABCA4 and two variants of established pathogenicity: p.N965S and p.C1488R. Our results were consistent with previous reports and clinical phenotypes. We also applied this platform to characterize the VUS p.Y1779F and observed a functional impairment, suggesting a potential pathogenic impact. This approach offers an efficient, high-throughput method for ABCA4 VUS characterization. Our research points to the significant promise of the VLP-based system in the functional analysis of membrane proteins, offering important perspectives on the diseasecausing potential of genetic variants and shedding light on genetic conditions involving such proteins. [ABSTRACT FROM AUTHOR]

Virus-like particles (VLPs) are nanoparticles that are self-assembled from one or more structural proteins, which can be arranged in several layers or contain a lipid outer membrane. Due to the lack of genetic material, VLPs cannot infect host cells, but are highly immunogenic and can induce immune responses different from conventional inactivated vaccines. VLPs can be produced using a variety of systems including bacterial, yeast, plant, insect and mammalian cells. Compared with traditional vaccines, VLPs have incomparable advantages, so they are becoming more and more popular in the biomedical field. To date, a series of vaccine candidates based on VLPs have been developed for immunization and prevention of various infectious diseases. At the same time, the recent successful application of VLPs in targeted drug delivery and gene therapy has attracted attention. This paper mainly reviews the commonly used expression systems of VLPs and the research progress of their applications. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Virus-like particles (VLPs) have been studied and used as vaccines to control foot-and-mouth disease (FMD). Mast cells (MCs) express various pattern recognition receptors that recognize pathogens and secrete numerous cytokines to initiate and modulate immune responses. Our previous study showed that bone marrow-derived mast cells (BMMCs) can recognize foot-and-mouth disease virus-like particles (FMDV-VLPs) to differentially express various cytokines and that histone acetylation can regulate the cytokines secreted during BMMC recognition of FMDV-VLPs. To demonstrate the role of DNA methylation in this response process, BMMCs that recognize FMDV-VLPs were treated with azacytidine (5-AZA), an inhibitor of DNA methylation transferase. We prepared FMDV-VLPs as described previously and cultured the BMMCs. The transcription and expression of key cytokines and transcription factors were determined using real-time quantitative PCR (RT-qPCR) and Western blotting. Results showed that pre-treatment with AZA resulted in the increased transcription and expression of tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-13, and IL-10, while the changes in IL-13 transcription and IL-6 expression were irrelevant to mannose receptors (MRs). Furthermore, analysis of the transcription factors indicated that both the transcription and expression of nuclear factor-kappa B (NF-κB) increased significantly in the AZA pre-treated group, indicating that DNA methylation may also regulate NF-κB expression to modulate TNF-α, IL-13, and IL-6. However, pre-treatment with AZA did not alter the expression of microphthalmia-associated transcription factor (MITF) or GATA-2. All the data demonstrate that DNA methylation negatively regulates the transcription and expression of TNF-α, IL-13, IL-10, and IL-6 secreted by recognizing FMDV-VLPs. These results provide new ideas for the mast cell-based design of more effective vaccine adjuvants and targeted therapies in the future. [ABSTRACT FROM AUTHOR]

The article discusses the development of a trivalent SARS-CoV-2 virus-like particle vaccine, RBDV14 M, targeting newly emerged variants such as XBB.1, BQ.1.1, EG.5, and BA.2.86. The vaccine induced superior antibody responses and provided protection against these variants in preclinical studies. The study highlights the potential of RBDV14 M as a next-generation SARS-CoV-2 vaccine candidate, demonstrating broad-spectrum neutralization and protection against specific variants. [Extracted from the article]

The continued development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has the potential to greatly impact clinical medicine, particularly for disease diagnosis and treatment. Despite high demand for the in vivo delivery of CRISPR-based therapies, significant challenges persist. These include rapid degradation by enzymes, inefficient disease site targeting, and the risk of undesired off-target outcomes. Nanoparticulate platforms, with their tailorable properties, have been engineered to efficiently package CRISPR payloads in various formats, including as plasmid DNA, mRNA, and ribonucleoprotein complexes, for in vivo delivery. Among them, recombinant adeno-associated viruses, virus-like particles, and lipid nanoparticles have displayed exceptional promise. This review will discuss the development of these and other nanocarriers for in vivo CRISPR-based genome editing. [ABSTRACT FROM AUTHOR]

Background/Objectives: The impact of virion maturation on neutralizing antibody responses in HIV treatment is not fully understood. This study examines whether antiretroviral regimens (ART) with boosted protease inhibitors (b-PI), which increase exposure to immature virions, affect neutralization capacity compared to Non-b-PI regimens. Methods: Neutralization activity was assessed in 45 HIV-infected individuals on b-PI regimens and 56 on Non-b-PI regimens, adjusting for factors like infection duration, ART initiation, and immune markers. Individuals on b-PI regimens had significantly lower neutralization scores [mean: 6.1, 95% Confidence Interval (CI): 5.3–6.9] than those on Non-b-PI regimens (mean: 8.9, 95% CI: 8.0–9.9; p < 0.0001). This difference was not explained by infection duration or CD4+ counts. CD4+/CD8+ ratios were positively associated with neutralization, while b-PI use was negatively associated. A regression model indicated that b-PI use significantly predicted lower neutralization scores (beta = −0.30, p = 0.049). Conclusions: These findings suggest that exposure to immature virions via b-PI use reduces neutralizing antibody responses, highlighting the importance of virion maturation in antibody induction. ART regimens promoting exposure to mature virions may enhance neutralization, with potential implications for HIV vaccine design. Further research is needed to explore implications for HIV vaccine design, especially using virus-like particles. [ABSTRACT FROM AUTHOR]

Simple Summary: Insect bite hypersensitivity (IBH) is a common skin allergic condition in horses caused by insect bites, mainly of Culiocides species. Affected horses develop severe itchy lesions, up to traumatic skin injuries. Eosinophils are known to play a crucial role in the pathogenesis of IBH. Recently, we described two subsets of eosinophils: inflammatory eosinophils (iEos) dominant in blood of IBH-affected horses, and resident eosinophils (rEos) present in blood of healthy horses. iEos and rEos were distinguishable by size, granularity, and proteins present on their cell surface. Interleukin (IL)-5, the main activator and regulator of eosinophils, is our vaccine target. Vaccinated horses showed a significant reduction of total eosinophils, in particular iEos, where the very few remaining eosinophils still showed iEos phenotype. In the present study, we followed the phenotype of eosinophil subsets in the 2nd year of vaccination in IBH-affected horses. Our results showed comparably lower levels of iEos and a significant increase of rEos in 2nd compared to 1st year vaccinated and unvaccinated horses. This suggests a shift from iEos to the rEos phenotype, the dominant eosinophil type of healthy horses. The change in size, granularity and migration properties suggests a benefit of long term vaccination of IBH-affected horses. 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. [ABSTRACT FROM AUTHOR]

Abstract Background The garden dormouse (Eliomys quercinus) has experienced a significant population decline across Europe in recent decades. While habitat loss and climate change are often cited as primary factors, pathogen exposure, either to novel or to previously known, may play a role in such a decline. This study aimed to investigate the presence of polyomaviruses in garden dormice, given that these viruses are highly prevalent and can cause disease, particularly in immunocompromised individuals. Methods The carcasses of garden dormice (n = 89) were collected throughout Germany. Kidney samples were tested for the presence of polyomavirus DNA using nested degenerate and specific diagnostic PCRs. Seroprevalence was assessed from chest cavity fluid samples through an enzyme-linked immunosorbent assay using polyomavirus VP1 virus-like particles produced in yeast. Results A new polyomavirus, related to chimpanzee (Pan troglodytes) polyomaviruses 4 and 5 and human Merkel cell polyomavirus, was identified in the garden dormouse. Two 5,380 bp-length complete viral genomes were sequenced from dormice kidney samples (sequences PQ246041 and PQ246042). Genes encoding the putative structural proteins VP1, VP2, and VP3, as well as the Large, Middle, and small T antigens, containing conserved functional domains were identified. Polyomavirus DNA was detected in 2 of 74 dormice (2.7%, 95% confidence interval: 0-6.4%) through PCR, while 12 of 69 animals (17.4%, 95% confidence interval: 8.4-26.3%) tested positive for polyomavirus-specific antibodies. Conclusions In conclusion, here we describe a novel polyomavirus in the garden dormouse with molecular and serological detection. Pairwise sequence comparison and phylogenetic analysis suggest that this novel virus may represent a novel species within the genus Alphapolyomavirus. Future work should examine if this virus is garden dormouse-specific and whether it is associated with disease in dormice.

Abstract Cervical cancer is the fourth most prevalent cancer among women globally, with Thai women ranking it as the third most common. At present, a prophylactic vaccine, containing virus-like particles (VLPs) of HPV L1 capsid protein, is widely recognized as one of the major prevention strategies for cervical cancer. Unfortunately, due to a low cross-protection among subtypes, protection against each HPV subtype requires vaccination with VLPs of that specific subtype. This study aimed to optimize the cultivation medium and conditions to maximize the volumetric yield of HPV52 L1 protein produced by the recombinant H. polymorpha HPV52. The results revealed that supplementation of a complex organic nitrogen source HySoy (at 10 g/L) into SYN6 medium resulted in a significantly higher specific HPV52 L1 yield and the maximum specific and volumetric HPV52 L1 protein yield were obtained at 30℃. In this study, the volumetric yield of HPV52 L1 could be increased from 50.9 mg/L in flask-scale cultivation to 2728 mg/L in High Cell Density Cultivation or HCDC (53-folds) with a significant improvement in terms of productivity, from 0.85 mg/L.h in flask-scale cultivation to 22.7 mg/L.h in HCDC (27-folds).

Abstract Humans get SARS-CoV-2 infection mainly through inhalation; thus, vaccine that induces protective immunity at the virus entry site is important for early control of the infection. In this study, two anionic liposome (L)-adjuvanted VLP vaccines against SARS-CoV-2 were formulated. Baculovirus-Sf21 insect cell system was used for production of VLPs made of full-length S, M and E proteins. S protein of one vaccine (L-SME-VLPs) contained furin cleavage site at the S1/S2 junction, while that of another vaccine (L-S′ME-VLPs) did not. Both vaccines were innocuous and immunogenic when administered IP and IN to mice. Mice immunized IP with L-SME-VLPs/L-S′ME-VLPs (three doses, two-weeks intervals) had serum virus neutralizing (VN) antibodies (in falling order of isotype frequency): IgG3, IgA and IgG2a/IgG3, IgA and IgM, respectively. The L-S′ME VLPs vaccine induced significantly higher serum VN antibody titers than the L-SME-VLPs vaccine. All mice immunized IN with both vaccines had significant rise of VN antibodies in their bronchoalveolar lavage fluids (BALF). The VN antibodies in 67% of immunized mice were Th1- isotypes (IgG2a and/or IgG2b); the immunized mice had also other antibody isotypes in BALF. The intranasal L-S′ME-VLPs should be tested further step-by-step towards the clinical use as effective and safe vaccine against SARS-CoV-2.

This article provides a comprehensive overview of research focusing on the role of antibodies, cytokines, complement proteins, major histocompatibility complex (MHC) molecules, and Toll-like receptors (TLRs) in the immune response and their potential as targets for immunotherapy. The review specifically examines the influence of various carriers on the immune activity of proteins, with a particular emphasis on the role of carriers in developing therapeutic approaches for diseases including cancer, autoimmune disorders, and infections. The findings highlight the importance of understanding the molecular mechanisms underlying the immune response and the role of different components of the immune system. Antibodies, as key components of adaptive immunity, play a crucial role in pathogen neutralization and can be utilized as targets for immunotherapy. Cytokines and complement proteins serve multiple functions, including immune cell activation, antiviral activity, and regulation of inflammatory processes. MHC molecules facilitate antigen presentation and activation of adaptive immunity. TLRs recognize pathogen-associated molecular patterns and initiate the immune response. Current research has also demonstrated the potential of lipid-based carriers, proteins, carbohydrates, and nucleic acids for enhancing the immune activity of proteins. The review discusses the use of carriers to improve the immune activity of proteins, which can be valuable for developing new vaccines and therapeutic agents. In recent years, there has been increasing interest in proteinbased therapeutic approaches, including monoclonal antibodies, cytokines, and others. The efficacy of these methods is influenced by the choice of carrier molecule. Conjugation of proteins with other molecules such as nanoparticles or liposomes can enhance stability, specificity, and efficacy. The presence of carriers on the surface of tumor cells can stimulate anti-tumor immune responses. However, challenges remain in the development of carrier-based therapies including potential carrier-induced immunogenicity, which may trigger undesired immune responses and limit therapeutic efficacy. Additionally, the complex selection of appropriate protein carriers for specific therapeutic applications requires further investigation into the underlying mechanisms of carrier function and immune activation. As based on the analysis of scientific literature, this review establishes that the use of carriers and ligands represents a promising approach for enhancing protein immune activity and developing new vaccination and immunotherapy strategies.

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.

Abstract Picornaviridae are non-enveloped ssRNA viruses that cause diseases such as poliomyelitis, hand-foot-and-mouth disease (HFMD), hepatitis A, encephalitis, myocarditis, and foot-and-mouth disease (FMD). Virus-like particles (VLPs) vaccines mainly comprise particles formed through the self-assembly of viral capsid proteins (for enveloped viruses, envelope proteins are also an option). They do not contain the viral genome. On the other hand, the nanoparticles vaccine (NPs) is mainly composed of self-assembling biological proteins or nanomaterials, with viral antigens displayed on the surface. The presentation of viral antigens on these particles in a repetitive array can elicit a strong immune response in animals. VLPs and NPs can be powerful platforms for multivalent antigen presentation. This review summarises the development of virus-like particle vaccines (VLPs) and nanoparticle vaccines (NPs) against picornaviruses. By detailing the progress made in the fight against various picornaviruses such as poliovirus (PV), foot-and-mouth disease virus (FMDV), enterovirus (EV), Senecavirus A (SVA), and encephalomyocarditis virus (EMCV), we in turn highlight the significant strides made in vaccine technology. These advancements include diverse construction methods, expression systems, elicited immune responses, and the use of various adjuvants. We see promising prospects for the continued development and optimisation of VLPs and NPs vaccines. Future research should focus on enhancing these vaccines' immunogenicity, stability, and delivery methods. Moreover, expanding our understanding of the interplay between these vaccines and the immune system will be crucial. We hope these insights will inspire and guide fellow researchers in the ongoing quest to combat picornavirus infections more effectively.

Recombinant technology-based vaccines have emerged as a highly effective way to prevent a wide range of illnesses. The technology improved vaccine manufacturing, rendering it more efficient and economical. These vaccines have multiple advantages compared to conventional vaccines. The pandemic has heightened awareness of the advantages of these vaccine technologies; trust and acceptance of these vaccines are steadily growing globally. This work offers an overview of the prospects and advantages associated with recombinant vaccines. Additionally, it discusses some of the challenges likely to arise in the future. Their ability to target diverse pathogen classes underscores their contributions to preventing previously untreatable diseases (especially vector-borne and emerging diseases) and hurdles faced throughout the vaccine development process, especially in enhancing the effectiveness of these vaccines. Moreover, their compatibility with emerging vaccination platforms of the future like virus-like particles and CRISPR/Cas9 for the production of next-generation vaccines may offer many prospects. This review also reviewed the hurdles faced throughout the vaccine development process, especially in enhancing the effectiveness of these vaccines against vector-borne diseases, emerging diseases, and untreatable diseases with high mortality rates like AIDS as well as cancer.

Canan Bayraktar,1,&ast; Alisan Kayabolen,1,&ast; Arda Odabas,1 Aysegul Durgun,1 Ipek Kok,1 Kenan Sevinc,1 Aroon Supramaniam,2 Adi Idris,2,3 Tugba Bagci-Onder1 1Koç University Research Center for Translational Medicine (KUTTAM), Koç University, Istanbul, Turkey; 2Menzies Health Institute Queensland, School of Medical Science Griffith University, Gold Coast Campus, Brisbane, QLD, Australia; 3Centre for Immunology and Infection Control, School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia&ast;These authors contributed equally to this workCorrespondence: Tugba Bagci-Onder; Alisan Kayabolen, Email tuonder@ku.edu.tr; akayabol@mit.eduPurpose: Over the past three years, extensive research has been dedicated to understanding and combating COVID-19. Targeting the interaction between the SARS-CoV-2 Spike protein and the ACE2 receptor has emerged as a promising therapeutic strategy against SARS-CoV-2. This study aimed to develop ACE2-coated virus-like particles (ACE2-VLPs), which can be utilized to prevent viral entry into host cells and efficiently neutralize the virus.Methods: Virus-like particles were generated through the utilization of a packaging plasmid in conjunction with a plasmid containing the ACE2 envelope sequence. Subsequently, ACE2-VLPs and ACE2-EVs were purified via ultracentrifugation. The quantification of VLPs was validated through multiple methods, including Nanosight 3000, TEM imaging, and Western blot analysis. Various packaging systems were explored to optimize the ACE2-VLP configuration for enhanced neutralization capabilities. The evaluation of neutralization effectiveness was conducted using pseudoviruses bearing different spike protein variants. Furthermore, the study assessed the neutralization potential against the Omicron BA.1 variant in Vero E6 cells.Results: ACE2-VLPs showed a high neutralization capacity even at low doses and demonstrated superior efficacy in in vitro pseudoviral assays compared to extracellular vesicles carrying ACE2. ACE2-VLPs remained stable under various environmental temperatures and effectively blocked all tested variants of concern in vitro. Notably, they exhibited significant neutralization against Omicron BA.1 variant in Vero E6 cells. Given their superior efficacy compared to extracellular vesicles and proven success against live virus, ACE2-VLPs stand out as crucial candidates for treating SARS-CoV-2 infections.Conclusion: This novel therapeutic approach of coating VLPs with receptor particles provides a proof-of-concept for designing effective neutralization strategies for other viral diseases in the future.Keywords: ACE2, virus like particles, SARS-CoV-2, neutralization, VLP, neutralization, escape mutations, spike protein

Picornaviridae are non-enveloped ssRNA viruses that cause diseases such as poliomyelitis, hand-foot-and-mouth disease (HFMD), hepatitis A, encephalitis, myocarditis, and foot-and-mouth disease (FMD). Virus-like particles (VLPs) vaccines mainly comprise particles formed through the self-assembly of viral capsid proteins (for enveloped viruses, envelope proteins are also an option). They do not contain the viral genome. On the other hand, the nanoparticles vaccine (NPs) is mainly composed of self-assembling biological proteins or nanomaterials, with viral antigens displayed on the surface. The presentation of viral antigens on these particles in a repetitive array can elicit a strong immune response in animals. VLPs and NPs can be powerful platforms for multivalent antigen presentation. This review summarises the development of virus-like particle vaccines (VLPs) and nanoparticle vaccines (NPs) against picornaviruses. By detailing the progress made in the fight against various picornaviruses such as poliovirus (PV), foot-and-mouth disease virus (FMDV), enterovirus (EV), Senecavirus A (SVA), and encephalomyocarditis virus (EMCV), we in turn highlight the significant strides made in vaccine technology. These advancements include diverse construction methods, expression systems, elicited immune responses, and the use of various adjuvants. We see promising prospects for the continued development and optimisation of VLPs and NPs vaccines. Future research should focus on enhancing these vaccines' immunogenicity, stability, and delivery methods. Moreover, expanding our understanding of the interplay between these vaccines and the immune system will be crucial. We hope these insights will inspire and guide fellow researchers in the ongoing quest to combat picornavirus infections more effectively. [ABSTRACT FROM AUTHOR]

The poultry industry, a cornerstone of global food security, faces dynamic challenges exacerbated by viral diseases. This review traces the trajectory of poultry vaccination, evolving from traditional methods to the forefront of innovation Virus-Like Particle (VLP) vaccines. Vaccination has been pivotal in disease control, but traditional vaccines exhibit some limitations. This review examines the emergence of VLPs as a game-changer in poultry vaccination. VLPs, mimicking viruses without replication, offer a safer, targeted alternative with enhanced immunogenicity. The narrative encompasses VLP design principles, production methods, immunogenicity, and efficacy against major poultry viruses. Challenges and prospects are explored, presenting VLP vaccines as a transformative technique in poultry disease control. Understanding their potential empowers industry stakeholders to navigate poultry health management with precision, promising improved welfare, reduced economic losses, and heightened food safety. [ABSTRACT FROM AUTHOR]

The capsid domain of the Gag protein from the human endogenous retrovirus HML‐2 can form virus‐like particles (VLPs) in vitro. Such particles made from proteins from other organisms are used as nanocarriers. Repeated use of exogeneous VLPs may however lead to an immune response. Recombinantly produced and purified capsid domain from HML‐2 could, given its human origin, provide a nanocarrier system, which potentially could evade an immune response. It is however not known if capsids formed in vitro by the HML‐2 Gag capsid domain can be loaded with a molecular cargo and if such particles are stable. In this work, we investigated how the assembly efficiency change with conditions. We demonstrate that once formed the capsids are stable over a much broader range of pH than the range of pH that allows capsid formation, and that the capsids are stable for weeks. We furthermore show that HML‐2 capsids can encapsulate small molecules. Our results suggest that the HML‐2 capsids potentially may serve as stable nanocarriers protecting its cargo from the surrounding environment. [ABSTRACT FROM AUTHOR]

In this work, we introduce a 3D-printable virus-like particle (VLP)-enhanced cross-linked biopolymer system. VLPs displaying surface-available acrylate groups were prepared through aza-Michael addition to serve as resins. The VLP resins were then photopolymerized into a poly(ethylene glycol) diacrylate (PEGDA) network following DLP 3D printing. This approach represents a convergence of disciplines, where the synergistic interaction between virology and additive manufacturing unlocks new frontiers in biotechnology. [ABSTRACT FROM AUTHOR]

Virus-like particles (VLPs) are used as nanocontainers for targeted drug, protein, and vaccine delivery. The phage P22 VLP is an ideal macromolecule delivery vehicle, as it has a large exterior surface area, which facilitates multivalent genetic and chemical modifications for cell recognition and penetration. Arginine-rich cell-penetrating peptides (CPPs) can increase cargo transport efficiency in vivo. However, studies on the tissue distribution and retention of P22 VLPs mediated by TAT and 8R are lacking. This study aimed to analyze the TAT and 8R effects on the P22 VLPs transport efficiency and tissue distribution both in vitro and in vivo. We used a prokaryotic system to prepare P22 VLP self-assembled particles and expressed TAT-or 8R-conjugated mCherry on the VLP capsid protein as model cargoes and revealed that the level of P22 VLP-mCherry penetrating the cell membrane was low. However, both TAT and 8R significantly promoted the cellular uptake efficiency of P22 VLPs in vitro, as well as enhanced the tissue accumulation and retention of P22 VLPs in vivo. At 24 h postinjection, TAT enhanced the tissue distribution and retention in the lung, whereas 8R could be better accumulation in brain. Thus, TAT was superior in terms of cellular uptake and tissue accumulation in the P22 VLPs delivery system. Understanding CPP biocompatibility and tissue retention will expand their potential applications in macromolecular cargo delivery. [ABSTRACT FROM AUTHOR]

Tumor neoantigen peptide vaccines hold potential for boosting cancer immunotherapy, yet efficiently co-delivering peptides and adjuvants to antigen-presenting cells in vivo remains challenging. Virus-like particle (VLP), which is a kind of multiprotein structure organized as virus, can deliver therapeutic substances into cells and stimulate immune response. However, the weak targeted delivery of VLP in vivo and its susceptibility to neutralization by antibodies hinder their clinical applications. Here, we first designed a novel protein carrier using the mammalian-derived capsid protein PEG10, which can self-assemble into endogenous VLP (eVLP) with high protein loading and transfection efficiency. Then, an engineered tumor vaccine, named ePAC, was developed by packaging genetically encoded neoantigen into eVLP with further modification of CpG-ODN on its surface to serve as an adjuvant and targeting unit to dendritic cells (DCs). Significantly, ePAC can efficiently target and transport neoantigens to DCs, and promote DCs maturation to induce neoantigen-specific T cells. Moreover, in mouse orthotopic liver cancer and humanized mouse tumor models, ePAC combined with anti-TIM-3 exhibited remarkable antitumor efficacy. Overall, these results support that ePAC could be safely utilized as cancer vaccines for antitumor therapy, showing significant potential for clinical translation. [ABSTRACT FROM AUTHOR]

Human noroviruses (HuNoVs) are a leading cause of acute viral gastroenteritis worldwide. Infectious outbreaks due to recombinant NoV genotype called GII.P16-GII.2 have been frequently reported since 2016. In this study, we expressed the major capsid protein VP1 from three GII.2 NoV strains using the recombinant baculovirus expression system. The assembly, histo-blood group antigen (HBGA)-binding patterns, and cross-blocking abilities of VP1 proteins were investigated. All the three NoV VP1 proteins successfully assembled into virus-like particles (VLPs). The HBGA-binding assay demonstrated a temporal binding pattern. The latest isolate bound to saliva samples of all blood types. Sequence alignment suggested that the observed gain in HBGA-binding ability was attributed to a limited number of amino acid mutations. Using chimeric VP1 proteins, we demonstrated that synergistic effects resulted in enhanced binding ability. Bile salts increased GII.2 VLP avidity for HBGAs except GII.2-2011/M1. In vitro blockade assay of salivary HBGA-VLP binding demonstrated the presence of cross-blocking effects among different strains. This study provides insight into the evolutionary binding characteristics and cross-blocking effects of GII.2 NoVs to facilitate the development of measures to control this type of viruses. [ABSTRACT FROM AUTHOR]