Virus-like particles (VLP)

Malaria, which is caused by the Plasmodium parasite and transmitted by mosquitoes, continues to be a major global health issue. The worldwide health community continues to work toward finding a conclusive answer to the malaria problem, but it is still elusive. Developing a successful malaria vaccine has proven difficult due to the Plasmodium parasite's complicated life cycle and ability to change and develop resistance to interventions rapidly. Amidst this backdrop, the advent of mRNA Lipid Nanoparticle (mRNA-LNP) vaccines, exemplified by their resounding success in mitigating the Coronavirus Disease 2019 (COVID-19) pandemic, has kindled newfound hope in vaccine development. This review examines the potential of leveraging mRNA technology to induce a robust immune response, thereby potentially revolutionising the landscape of malaria prevention through the development of breakthrough malaria vaccines. The intricate interplay between the efficacy of the mRNA-LNP vaccine against COVID-19 and its prospective utility in addressing malaria is also deliberated upon.

Virus-based nanoparticles constitute a promising platform for the creation of efficient vaccines and nanomaterials. Previously we demonstrated, that the recombinant tail tube protein gp39 of vB_EcoS_NBD2 bacteriophage self-assembles into extremely long (from 0.1 to >3.95 μm), flexible, and stable polytubes when produced in Saccharomyces cerevisiae. To develop a tubular platform for multivalent display of foreign antigens, yeast-derived recombinant tail tube protein gp39 was chosen as a scaffold. The carboxy-terminal fusions of gp39 with various antigens up to 238 amino acids in length resulted in different synthesis efficiency and self-assembly capacity. Recombinant gp39 fused with green fluorescent protein (eGFP) comprising 238 amino acid residues was capable to self-assemble into short fluorescent polytubes with retained eGFP functional activity. By demonstrating the display of active foreign antigens on the exterior surface of polytubes, these structures may provide a promising tool for diverse applications in nanotechnology.

Virus-like particles (VLPs) are highly organized particles that self-assemble from viral structural proteins. Like parental viruses, VLPs can be either non-enveloped or enveloped and can be produced in different expression systems depending on their complexity. Over the last three decades, VLPs have developed as a high-priority alternative to traditional vaccines against infectious pathogens due to their safety, simplicity and favorable immunological characteristics to induce both humoral and cellular immune responses. Most of emerging and re-emerging viruses that pose a continuous threat to human health are enveloped, but few vaccines are currently available. Advances in expression technology for complex, enveloped VLPs provide new possibilities to develop potent vaccines against pathogenic enveloped viruses. This review describes major progress and challenges in the production of enveloped VLPs, with respect to the main principles in the assembly and budding process, factors that need to be taken into account for the design strategies and choice of relevant production platforms.

Subunit vaccines containing one or more target antigens from pathogenic organisms represent safer alternatives to whole pathogen vaccines. However, the antigens by themselves are not sufficiently immunogenic and require additives known as adjuvants to enhance immunogenicity and protective efficacy. Assembly of the antigens into virus-like nanoparticles (VLPs) is a better approach as it allows presentation of the epitopes in a more native context. The repetitive, symmetrical, and high density display of antigens on the VLPs mimic pathogen-associated molecular patterns seen on bacteria and viruses. The antigens, thus, might be better presented to stimulate host's innate as well as adaptive immune systems thereby eliciting both humoral and cellular immune responses. Bacteriophages such as phage T4 provide excellent platforms to generate the nanoparticle vaccines. The T4 capsid containing two non-essential outer proteins Soc and Hoc allow high density array of antigen epitopes in the form of peptides, domains, full-length proteins, or even multi-subunit complexes. Co-delivery of DNAs, targeting molecules, and/or molecular adjuvants provides additional advantages. Recent studies demonstrate that the phage T4 VLPs are highly immunogenic, do not need an adjuvant, and provide complete protection against bacterial and viral pathogens. Thus, phage T4 could potentially be developed as a "universal" VLP platform to design future multivalent vaccines against complex and emerging pathogens.

Catherine Cleuziat, Boehringer-Ingelheim, France catherine.cleuziat@boehringer-ingelheim.com Sophie Biard, Boehringer-Ingelheim, France Géraldine Popovic, Boehringer-Ingelheim, France Sylvain Lagresle, Boehringer-Ingelheim, France Chloé Damiany, Boehringer-Ingelheim, France Christine Coupier, Boehringer-Ingelheim, France Chun Fang Shen, NRC, USA Amine Kamen, MacGill University, Canada Hervé Poulet, Boehringer-Ingelheim, France Noël Detraz, Boehringer-Ingelheim, France Zahia Hannas, Boehringer-Ingelheim, France

Different strategies are being worked out for engineering the original baculovirus expression vector (BEV) system to produce cost-effective clinical biologics at commercial scale. To date, thousands of highly variable molecules in the form of heterologous proteins, virus-like particles, surface display proteins/antigen carriers, heterologous viral vectors and gene delivery vehicles have been produced using this system. These products are being used in vaccine production, tissue engineering, stem cell transduction, viral vector production, gene therapy, cancer treatment and development of biosensors. Recombinant proteins that are expressed and post-translationally modified using this system are also suitable for functional, crystallographic studies, microarray and drug discovery-based applications. Till now, four BEV-based commercial products (Cervarix ® , Provenge ® , Glybera ® and Flublok ®) have been approved for humans, and myriad of others are in different stages of preclinical or clinical trials. Five products (Porcilis ® Pesti, BAYOVAC CSF E2 ® , Circumvent ® PCV, Ingelvac CircoFLEX ® and Porcilis ® PCV) got approval for veterinary use, and many more are in the pipeline. In the present chapter, we have emphasized on both approved and other baculovirus-based products produced in insect cells or larvae that are important from clinical perspective and are being developed as preventive, diagnostic or therapeutic agents. Further, the potential of recombinant adeno-associated virus (rAAV) as gene delivery vector has been described. This system, due to its relatively extended gene expression, lack of pathogenicity and the ability to transduce

Subunit vaccines containing one or more target antigens from pathogenic organisms represent safer alternatives to whole pathogen vaccines. However, the antigens by themselves are not sufficiently immunogenic and require additives known as adjuvants to enhance immunogenicity and protective efficacy. Assembly of the antigens into virus-like nanoparticles (VLPs) is a better approach as it allows presentation of the epitopes in a more native context. The repetitive, symmetrical, and high density display of antigens on the VLPs mimic pathogen-associated molecular patterns seen on bacteria and viruses. The antigens, thus, might be better presented to stimulate host's innate as well as adaptive immune systems thereby eliciting both humoral and cellular immune responses. Bacteriophages such as phage T4 provide excellent platforms to generate the nanoparticle vaccines. The T4 capsid containing two non-essential outer proteins Soc and Hoc allow high density array of antigen epitopes in the form of peptides, domains, full-length proteins, or even multi-subunit complexes. Co-delivery of DNAs, targeting molecules, and/or molecular adjuvants provides additional advantages. Recent studies demonstrate that the phage T4 VLPs are highly immunogenic, do not need an adjuvant, and provide complete protection against bacterial and viral pathogens. Thus, phage T4 could potentially be developed as a "universal" VLP platform to design future multivalent vaccines against complex and emerging pathogens.

Background Leading transmission-blocking vaccine candidates such as Plasmodium falciparum surface protein 25 (Pfs25 gene) may undergo antigenic alterations which may render them ineffective or allele-specific. This study examines the level of genetic diversity, signature of selection and drivers of Pfs25 polymorphisms of parasites population in regions of western Kenya with varying malaria transmission intensities. Methods Dry blood spots (DBS) were collected in 2018 and 2019 from febrile outpatients with malaria at health facilities in malaria-endemic areas of Homa Bay, Kisumu (Chulaimbo) and the epidemic-prone highland area of Kisii. Parasites DNA were extracted from DBS using Chelex method. Species identification was performed using real-time PCR. The 460 base pairs (domains 1-4) of the Pfs25 were amplified and sequenced for a total of 180 P. falciparuminfected blood samples. Results Nine of ten polymorphic sites were identified for the first time. Overall, Pfs25 exhibited low nucleotide diversity (0.04×10 −2) and low mutation frequencies (1.3% to 7.7%). Chulaimbo had the highest frequency (15.4%) of mutated sites followed by Kisii (6.7%) and Homa Bay (5.1%). Neutrality tests of Pfs25 variations showed significant negative values of Tajima's D (-2.15, p<0.01) and Fu's F (-10.91, p<0.001) statistics tests. Three loci pairs (123, 372), (364, 428) and (390, 394) were detected to be under linkage disequilibrium and none had

Background Leading transmission-blocking vaccine candidates such as Plasmodium falciparum surface protein 25 (Pfs25 gene) may undergo antigenic alterations which may render them ineffective or allele-specific. This study examines the level of genetic diversity, signature of selection and drivers of Pfs25 polymorphisms of parasites population in regions of western Kenya with varying malaria transmission intensities. Methods Dry blood spots (DBS) were collected in 2018 and 2019 from febrile outpatients with malaria at health facilities in malaria-endemic areas of Homa Bay, Kisumu (Chulaimbo) and the epidemic-prone highland area of Kisii. Parasites DNA were extracted from DBS using Chelex method. Species identification was performed using real-time PCR. The 460 base pairs (domains 1–4) of the Pfs25 were amplified and sequenced for a total of 180 P. falciparum-infected blood samples. Results Nine of ten polymorphic sites were identified for the first time. Overall, Pfs25 exhibited low n...

Chimeric virus-like particles (cVLPs) show great potential in improving public health as they are safe and effective vaccine candidates. The capsid protein of caliciviruses has been described previously as a self-assembling, highly immunogenic delivery platform. The ability to significantly induce cellular and humoral immunity can be used to boost the immune response to low immunogenic foreign antigens displayed on the surface of VLPs. Capsid proteins of caliciviruses despite sequence differences share similar architecture with structural loops that can be genetically modified to present foreign epitopes on the surface of cVLPs. Here, based on the VP1 protein of norovirus (NoV), we investigated the impact of the localization of the epitope in different structural loops of the P domain on the immunogenicity of the presented epitope. In this study, three distinct loops of NoV VP1 protein were genetically modified to present a multivalent influenza virus epitope consisting of a tandem ...

IntroductionThere is a growing demand for effective technologies for the delivery of antigen to antigen-presenting cells (APCs) and their immune-activation for the success of DNA vaccines. Therefore, dendritic cell (DC)-targeting T7 phages were used as a vehicle to deliver DNA vaccine.MethodsIn this study, a eukaryotic expression plasmid pEGFP-C1-HA2-AS containing the HA2 gene derived from the avian H5N1 virus and an anchor sequence (AS) gene required for the T7 phage packaging process was developed. To verify the feasibility of phage delivery, the plasmid encapsulated in DC-targeting phage capsid through the recognition of AS was evaluated both in vitro and in vivo. The pEGFP-C1-HA2-AS plasmid could evade digestion by DNase I by becoming encapsulated into the phage particles and efficiently expressed the HA2 antigen in DCs with the benefit of DC-targeting phages.ResultsFor chickens immunized with the DC-targeting phage 74 delivered DNA vaccine, the levels of IgY and IgA antibodies,...

The increasing demand for the characterization of large biomolecules such as monoclonal antibodies, double-stranded deoxyribonucleic acid (dsDNA), and virus-like particles (VLPs) is raising fundamental questions pertaining to their absorption (ingress) and escape (egress) kinetics from fully porous particles. The exact expression of their concentration profiles is derived as a function of time and radial position across a single sub-3 μm Bridge-Ethylene-Hybrid (BEH TM) Particle present in size exclusion chromatography (SEC) columns. The boundary condition at the external surface area of the particle is a rectangular concentration profile mimicking the passage of the chromatographic zone. Four different BEH Particles were considered in the calculations depending on the molecular size of the analyte: 2.0 μm 100 Å BEH Particles for small molecules, 2.0 μm 200 Å BEH Particles for monoclonal antibodies, 2.0 μm 300 Å BEH Particles for dsDNA (100 base pairs), and 2.5 μm 900 Å BEH Particles for virus-like particles (VLPs). The calculated concentration profiles of small molecules and monoclonal antibodies confirm that all BEH Particles present in the column reach quasi-instantaneously thermodynamic equilibrium with the bulk mobile phase during the passage of the chromatographic band. This is no longer the case for larger biomolecules such as dsDNA or VLPs, especially when the SEC particle is located near the column inlet and for high velocities. The kinetics of biomolecule egress is slower than its kinetics of ingress leading to pronounced peak tailing. The mean concentration of the largest biomolecules in the SEC particles remains always smaller than the maximum bulk concentration. This persistent and transient intra-particle diffusion regime has direct implications on the theoretical expressions of the observed retention factors and plate heights. Classical theories of chromatography assume uniform spatial distribution of the analyte in the particle volume: this hypothesis is not verified for the largest biomolecules. These results imply that non-porous particles or monolithic structures are the most promising stationary phases for the separation and purification of the largest biomolecules in life science.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Traditional veterinary virus vaccines, such as inactivated and live-attenuated vaccines, have achieved tremendous success in controlling many viral diseases of livestock and chickens worldwide. However, many recent viral outbreaks caused by different emerging and re-emerging viruses continue to be reported annually worldwide. It is therefore necessary to develop new control regimens. Nanoparticle research has received considerable attention in the last two decades as a promising platform with significant success in veterinary medicine, replacing traditional viral vector vaccines. However, the field of nanoparticle applications is still in its initial phase of growth. Here, we discuss various preparation methods, characteristics, physical properties, antiviral effects, and pharmacokinetics of well-developed nanoparticles and the potential of nanoparticles or nano-vaccines as a promising antiviral platform for veterinary medicine.

Background Leading transmission-blocking vaccine candidates such as Plasmodium falciparum surface protein 25 (Pfs25 gene) may undergo antigenic alterations which may render them ineffective or allele-specific. This study examines the level of genetic diversity, signature of selection and drivers of Pfs25 polymorphisms of parasites population in regions of western Kenya with varying malaria transmission intensities. Methods Dry blood spots (DBS) were collected in 2018 and 2019 from febrile outpatients with malaria at health facilities in malaria-endemic areas of Homa Bay, Kisumu (Chulaimbo) and the epidemic-prone highland area of Kisii. Parasites DNA were extracted from DBS using Chelex method. Species identification was performed using real-time PCR. The 460 base pairs (domains 1–4) of the Pfs25 were amplified and sequenced for a total of 180 P. falciparum-infected blood samples. Results Nine of ten polymorphic sites were identified for the first time. Overall, Pfs25 exhibited low n...

Africa bears the greatest burden of malaria with more than 200 million clinical cases and more than 600,000 deaths in 2020 alone. While malaria-associated deaths dropped steadily until 2015, the decline started to falter after 2016, highlighting the need for novel potent tools in the fight against malaria. Currently available tools, such as antimalarial drugs and insecticides are threatened by development of resistance by the parasite and the mosquito. The WHO has recently approved RTS,S as the first malaria vaccine for public health use. However, because the RTS,S vaccine has an efficacy of only 36% in young children, there is need for more efficacious vaccines. Indeed, based on the global goal of licensing a malaria vaccine with at least 75% efficacy by 2030, RTS,S is unlikely to be sufficient alone. However, recent years have seen tremendous progress in vaccine development. Although the COVID-19 pandemic impacted malaria control, the rapid progress in research towards the develop...

Virus-based nanoparticles constitute a promising platform for the creation of efficient vaccines and nanomaterials. Previously we demonstrated, that the recombinant tail tube protein gp39 of vB_EcoS_NBD2 bacteriophage self-assembles into extremely long (from 0.1 to >3.95 μm), flexible, and stable polytubes when produced in Saccharomyces cerevisiae. To develop a tubular platform for multivalent display of foreign antigens, yeast-derived recombinant tail tube protein gp39 was chosen as a scaffold. The carboxy-terminal fusions of gp39 with various antigens up to 238 amino acids in length resulted in different synthesis efficiency and self-assembly capacity. Recombinant gp39 fused with green fluorescent protein (eGFP) comprising 238 amino acid residues was capable to self-assemble into short fluorescent polytubes with retained eGFP functional activity. By demonstrating the display of active foreign antigens on the exterior surface of polytubes, these structures may provide a promising tool for diverse applications in nanotechnology.

Virus-like particles (VLPs) are virus-derived structures made up of one or more different molecules with the ability to self-assemble, mimicking the form and size of a virus particle but lacking the genetic material so they are not capable of infecting the host cell. Expression and self-assembly of the viral structural proteins can take place in various living or cell-free expression systems after which the viral structures can be assembled and reconstructed. VLPs are gaining in popularity in the field of preventive medicine and to date, a wide range of VLP-based candidate vaccines have been developed for immunization against various infectious agents, the latest of which is the vaccine against SARS-CoV-2, the efficacy of which is being evaluated. VLPs are highly immunogenic and are able to elicit both the antibody- and cell-mediated immune responses by pathways different from those elicited by conventional inactivated viral vaccines. However, there are still many challenges to this...

Porcine epidemic diarrhea virus (PEDV) is a member of the Alphacoronaviridae genus within the Coronaviridae family. It is the causative agent of porcine epidemic diarrhea, a disease that can have mortality rates as high as 100% in suckling piglets. PEDV causes severe economic loss, and has been in existence for decades. A panzootic starting in 2010 renewed interest in the development of a universal vaccine toward PEDV. This report details several design changes made to a Hepatitis B virus core antigen (HBcAg)-based recombinant vaccine strategy, and their effect in vivo. Initially, several multi-antigen vaccine candidates were able to elicit antibodies specific to three out of four B-cell epitopes inserted into the chimeric proteins. However, a lack of virus neutralization led to a redesign of the vaccines. The focus of the newly redesigned vaccines was to elicit a strong immune response to the YSNIGVCK amino acid motif from PEDV. Genetically modified new vaccine candidates were able to elicit a strong antibody (Ab) response to the YSNIGVCK epitope, which correlated with an increased ability to neutralize the CO strain of PEDV. Additionally, the location of the inserted PEDV epitopes within the vector protein was shown to affect the immune recognition toward the native HBcAg during vaccination.

Traditional veterinary virus vaccines, such as inactivated and live-attenuated vaccines, have achieved tremendous success in controlling many viral diseases of livestock and chickens worldwide. However, many recent viral outbreaks caused by different emerging and re-emerging viruses continue to be reported annually worldwide. It is therefore necessary to develop new control regimens. Nanoparticle research has received considerable attention in the last two decades as a promising platform with significant success in veterinary medicine, replacing traditional viral vector vaccines. However, the field of nanoparticle applications is still in its initial phase of growth. Here, we discuss various preparation methods, characteristics, physical properties, antiviral effects, and pharmacokinetics of well-developed nanoparticles and the potential of nanoparticles or nano-vaccines as a promising antiviral platform for veterinary medicine.

Virus-like particles (VLPs) have been shown to be strong activators of dendritic cells (DCs). DCs are the most potent antigen presenting cells (APCs) and their activation prompts the priming of immunity mediators based on B and T cells. The first step for the activation of DCs is the binding of VLPs to pattern recognition receptors (PRRs) on the surface of DCs, followed by VLP internalization. Like wild-type viruses, VLPs use specific PRRs from the DC; however, these recognition interactions between VLPs and PRRs from DCs have not been thoroughly reviewed. In this review, we focused on the interaction between proteins that form VLPs and PRRs from DCs. Several proteins that form VLP contain glycosylations that allow the direct interaction with PRRs sensing carbohydrates, prompting DC maturation and leading to the development of strong adaptive immune responses. We also discussed how the knowledge of the molecular interaction between VLPs and PRRs from DCs can lead to the smart design of VLPs, whether based on the fusion of foreign epitopes or their chemical conjugation, as well as other modifications that have been shown to induce a stronger adaptive immune response and protection against infectious pathogens of importance in human and veterinary medicine. Finally, we address the use of VLPs as tools against cancer and allergic diseases.

We report the crystal structure of the M2 ectodomain (M2e) in complex with a monoclonal antibody that binds the amino-terminus of M2. M2e stretches out into the antibody binding site to form an N-terminal β-turn near the bottom of the paratope. This M2e folding differs significantly from M2e in complex with an antibody that binds another part of M2e. This suggests that M2e can adopt at least two conformations that can elicit protective antibodies.

The first successful use of nanoparticles (NPs) for vaccination was reported almost 40 years ago with a virus-like particle-based vaccine against Hepatitis B. Since then, the term NP has been expanded to accommodate a large number of novel nano-sized particles engineered from a range of materials. The great interest in NPs is likely not only a result of the two successful vaccines against hepatitis B and Human Papilloma Virus (HPV) that use this technology, but also due to the versatility of those small-sized particles, as indicated by the wide range of applications reported so far, ranging from medicinal and cosmetics to purely technical applications. In this review, we will focus on the use of NPs, especially virus-like particles (VLPs), in the field of vaccines and will discuss their employment as vaccines, antigen display platforms, adjuvants and drug delivery systems.

Although electroporation has been widely accepted as the main gene transfer tool, there is still considerable scope to improve the electroporation efficiency of exogenous DNAs into primary cells. Here, we developed a square-wave pulsing protocol using OptiMEM-GlutaMAX for highly efficient transfection of murine embryonic fibroblasts (MEF) and induced pluripotency stem (iPS) cells using reporter genes as well as gRNA/Cas9-encoding plasmids. An electrotransfection efficiency of > 95% was achieved for both MEF and iPS cells using reporter-encoding plasmids. The protocol was efficient for plasmid sizes ranging from 6.2 to 13.5 kb. Inducing the error prone non-homologous end joining repair by gRNA/Cas9 plasmid transfection, a high rate of targeted gene knockouts of up to 98% was produced in transgenic cells carrying a single-copy of Venus reporter. Targeted deletions in the Venus transgene were efficiently (up to 67% deletion rate) performed by co-electroporation of two gRNAencoding plasmids. We introduced a plasmid electrotransfection protocol which is straightforward , cost-effective, and efficient for CRISPRing murine primary cells. This protocol is promising to make targeted genetic engineering using the CRISPR/Cas9 plasmid system. Abbreviations CRISPR Clustered regularly interspaced short palindromic repeats Cas9 CRISPR-associated protein 9 gRNA Guide RNA iPS Induced pluripotent stem cell KO Knockout MEF Mouse embryonic fibroblast RNPs Ribonucleoprotein The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) nuclease system is a straightforward , versatile, and highly efficient tool for genome editing of various organisms. Using 'all-in-one' expression vectors containing expression cassettes for guide RNAs and Cas9 nuclease/nickase it is possible to conduct CRISPR/Cas9 studies through a low-cost and straightforward approach 1. However, the efficiency of gene editing using plasmid-based delivery methods remains relatively low, which subsequently increased attention toward using alternate methods employing the Cas9 protein and/or guideRNA (gRNA), or using ribonucleoproteins (RNPs) 2,3. Basically, gene transfer into cells can be achieved via electroporation, lipofection, or viral transduction. Although viral gene transfer is very efficient, it requires time, skilled staff, and high levels of safety issues, whereas it has a limitation in the cargo size 4. On the other side, lipofection suffers from a low efficiency in primary cells. Electroporation is an approach to instantly create pores in the cell membrane using a burst electric pulse and to mediate the transfer of micro-and macro-molecules into cells, embryos, tissues, and organs 5. Since the early papers on electroporation 6 , it has been evident that gene transfer via electroporation is simple, easily applicable, and also efficient compared to lipofection 6. Although electroporation has been widely accepted as the main gene transfer tool, the underlying mechanism has not been completely understood 7. Therefore, optimization of various factors, such as the electroporation medium, cuvette type (path width, and length), as well as the pulsing method which includes the amount, number, duration, and interval of pulses is needed to have a high

Viral diseases, including avian influenza (AI) and Newcastle disease (ND), are an important cause of morbidity and mortality in poultry, resulting in significant economic losses. Despite the availability of commercial vaccines for the major viral diseases of poultry, these diseases continue to pose a significant risk to global food security. There are multiple factors for this: vaccine costs may be prohibitive, cold chain storage for attenuated live-virus vaccines may not be achievable, and commercial vaccines may protect poorly against local emerging strains. The development of transient gene expression systems in plants provides a versatile and robust tool to generate a high yield of recombinant proteins with superior speed while managing to achieve cost-efficient production. Plant-derived vaccines offer good stability and safety these include both subunit and virus-like particle (VLP) vaccines. VLPs offer potential benefits compared to currently available traditional vaccines, in...

Viral diseases, including avian influenza (AI) and Newcastle disease (ND), are an important cause of morbidity and mortality in poultry, resulting in significant economic losses. Despite the availability of commercial vaccines for the major viral diseases of poultry, these diseases continue to pose a significant risk to global food security. There are multiple factors for this: vaccine costs may be prohibitive, cold chain storage for attenuated live-virus vaccines may not be achievable, and commercial vaccines may protect poorly against local emerging strains. The development of transient gene expression systems in plants provides a versatile and robust tool to generate a high yield of recombinant proteins with superior speed while managing to achieve cost-efficient production. Plant-derived vaccines offer good stability and safety these include both subunit and virus-like particle (VLP) vaccines. VLPs offer potential benefits compared to currently available traditional vaccines, in...

The CRISPR-Cas9 technology represents a powerful tool for genome engineering in eukaryotic cells, advancing both fundamental research and therapeutic strategies. Despite the enormous potential of the technology, efficient and direct intracellular delivery of Cas9 ribonucleoprotein (RNP) complexes in target cells poses a significant hurdle, especially in refractive primary cells. In the present work, vapor nanobubble (VNB) photoporation was explored for Cas9 RNP transfection in a variety of cell types. Proof of concept was first demonstrated in H1299-EGFP cells, before proceeding to hard-to-transfect stem cells and T cells. Gene knockout levels over 80% and up to 60% were obtained for H1299 cells and mesenchymal stem cells, respectively. In these cell types, the unique possibility of VNB photoporation to knock out genes according to userdefined spatial patterns was demonstrated as well. Next, effective targeting of the programmed cell death 1 receptor and Wiskott-Aldrich syndrome gene in primary human T cells was demonstrated, reaching gene knockout levels of 25% and 34%, respectively. With a throughput of >200,000 T cells per second, VNB photoporation is a scalable and versatile intracellular delivery method that holds great promise for CRISPR-Cas9-mediated ex vivo engineering of cell therapy products.

The human genome is generally organized into stable chromosomes, and only tumor cells are known to accumulate kilobase (kb)-sized extrachromosomal circular DNA elements (eccDNAs). However, it must be expected that kb eccDNAs exist in normal cells as a result of mutations. Here, we purify and sequence eccDNAs from muscle and blood samples from 16 healthy men, detecting ~100,000 unique eccDNA types from 16 million nuclei. Half of these structures carry genes or gene fragments and the majority are smaller than 25 kb. Transcription from eccDNAs suggests that eccDNAs reside in nuclei and recurrence of certain eccDNAs in several individuals implies DNA circularization hotspots. Gene-rich chromosomes contribute to more eccDNAs per megabase and the most transcribed protein-coding gene in muscle, TTN (titin), provides the most eccDNAs per gene. Thus, somatic genomes are rich in chromosome-derived eccDNAs that may influence phenotypes through altered gene copy numbers and transcription of ful...

Chronic viral infections represent major challenges in contemporary medicine, virology and pharmacology. The virus-bearing hosts are commonly found in every parts of the world and it is extremely difficult to manage these patients. In addition, considerable numbers of these patients develop progressive diseases and severe complications. Finally, most of these patients act as permanent reservoirs of virus. Understandings of viral life cycle during the last decade of 20th century and the first decade of 21st century have allowed development of hundreds of antiviral agents for different diseases. But, the clinical efficacy of these drugs is not yet satisfactory. In addition, virologists have provided conclusive evidences suggesting that eradication of most chronic virus from infected hosts may an unachievable

In vivo genome editing represents a powerful strategy for both understanding basic biology and treating inherited diseases. However, it remains a challenge to develop universal and efficient in vivo genome-editing tools for tissues that comprise diverse cell types in either a dividing or non-dividing state. Here, we describe a versatile in vivo gene knock-in methodology that enables the targeting of a broad range of mutations and cell types through the insertion of a minigene at an intron of the target gene locus using an intracellularly linearized single homology arm donor. As a proof-of-concept, we focused on a mouse model of prematureaging caused by a dominant point mutation, which is difficult to repair using existing in vivo genome-editing tools. Systemic treatment using our new method ameliorated aging-associated phenotypes and extended animal lifespan, thus highlighting the potential of this methodology for a broad range of in vivo genome-editing applications.

We report the complete genome sequences of two isolates (RHDV-N11 and CBVal16) of variant rabbit hemorrhagic disease virus (RHDVb). Isolate N11 was detected in young domestic animals during a rabbit hemorrhagic disease (RHD) outbreak that occurred in 2011 on a rabbit farm in Navarra, Spain, while CBVal16 was isolated from a wild rabbit found dead in Valpaços, Northern Portugal, a year later. The viral sequences reported show 84.8-85.1 % and 78.3-78.5 % identity to RHDVAst/89 and RCV-A1 MIC-07, representative members of the pathogenic genogroup 1 RHDV and apathogenic rabbit calicivirus, respectively. In comparison with other RHDV isolates belonging to the previously known genogroups 1-6, RHDVb shows marked phenotypic differences, as it causes disease preferentially in young rabbits under 40 days of age and shows modified red blood cell agglutination profiles as well as antigenic differences that allow this variant to escape protection by the currently available vaccines.

Infectious and parasitic diseases have major negative economic and animal welfare impacts on aquaculture of salmonid species. Improved knowledge of the functional basis of host response and genetic resistance to these diseases is key to developing preventative and treatment options. Cell lines provide a valuable model to study infectious diseases in salmonids, and genome editing using CRISPR provides an exciting avenue to evaluate the function of specific genes in those systems. While CRISPR/Cas9 has been successfully performed in a Chinook salmon cell line (CHSE-214), there are no reports to date of editing of cell lines derived from the most commercially relevant salmonid species Atlantic salmon and rainbow trout, which are difficult to transduce and therefore edit using lentivirus-mediated methods. In the current study, a method of genome editing of salmonid cell lines using ribonucleoprotein (RNP) complexes was optimised and tested in the most commonly-used salmonid fish cell li...

SARS-CoV-2 has emerged as a major threat to global public health, resulting in global societal and economic disruptions. Here, we investigate the intramolecular and intermolecular RNA interactions of wildtype (WT) and a mutant (Δ382) SARS-CoV-2 virus in cells using high throughput structure probing on Illumina and Nanopore platforms. We identified twelve potentially functional structural elements within the SARS-CoV-2 genome, observed that identical sequences can fold into divergent structures on different subgenomic RNAs, and that WT and Δ382 virus genomes can fold differently. Proximity ligation sequencing experiments identified hundreds of intramolecular and intermolecular pair-wise interactions within the virus genome and between virus and host RNAs. SARS-CoV-2 binds strongly to mitochondrial and small nucleolar RNAs and is extensively 2’-O-methylated. 2’-O-methylation sites in the virus genome are enriched in the untranslated regions and are associated with increased pair-wise ...

Traditional veterinary virus vaccines, such as inactivated and live-attenuated vaccines, have achieved tremendous success in controlling many viral diseases of livestock and chickens worldwide. However, many recent viral outbreaks caused by different emerging and re-emerging viruses continue to be reported annually worldwide. It is therefore necessary to develop new control regimens. Nanoparticle research has received considerable attention in the last two decades as a promising platform with significant success in veterinary medicine, replacing traditional viral vector vaccines. However, the field of nanoparticle applications is still in its initial phase of growth. Here, we discuss various preparation methods, characteristics, physical properties, antiviral effects, and pharmacokinetics of well-developed nanoparticles and the potential of nanoparticles or nano-vaccines as a promising antiviral platform for veterinary medicine.

Traditional veterinary virus vaccines, such as inactivated and live-attenuated vaccines, have achieved tremendous success in controlling many viral diseases of livestock and chickens worldwide. However, many recent viral outbreaks caused by different emerging and re-emerging viruses continue to be reported annually worldwide. It is therefore necessary to develop new control regimens. Nanoparticle research has received considerable attention in the last two decades as a promising platform with significant success in veterinary medicine, replacing traditional viral vector vaccines. However, the field of nanoparticle applications is still in its initial phase of growth. Here, we discuss various preparation methods, characteristics, physical properties, antiviral effects, and pharmacokinetics of well-developed nanoparticles and the potential of nanoparticles or nano-vaccines as a promising antiviral platform for veterinary medicine.

Traditional veterinary virus vaccines, such as inactivated and live-attenuated vaccines, have achieved tremendous success in controlling many viral diseases of livestock and chickens worldwide. However, many recent viral outbreaks caused by different emerging and re-emerging viruses continue to be reported annually worldwide. It is therefore necessary to develop new control regimens. Nanoparticle research has received considerable attention in the last two decades as a promising platform with significant success in veterinary medicine, replacing traditional viral vector vaccines. However, the field of nanoparticle applications is still in its initial phase of growth. Here, we discuss various preparation methods, characteristics, physical properties, antiviral effects, and pharmacokinetics of well-developed nanoparticles and the potential of nanoparticles or nano-vaccines as a promising antiviral platform for veterinary medicine.

Trypanosomatids are divergent eukaryotes of high medical and economical relevance. Their biology exhibits original features which remain poorly understood; particularly Leishmania is known for its high degree of genomic plasticity which makes genomic manipulation challenging. CRISPR-Cas9 has been applied successfully to these parasites providing a robust tool to study non-essential gene functions. Here, we have developed a versatile inducible system combining Di-CRE recombinase and CRISPR-Cas9 advantages. Cas9 is used to integrate the LoxP sequences and the Cre-recombinase catalyzes the recombination between LoxP sites thereby excising the target gene. We used an L. mexicana cell line expressing Di-CRE, Cas9 and T7 polymerase and then transfected donor DNAs and single guide RNAs as PCR products. Since the location of LoxP sequences in the genomic DNA can interfere with the function and localization of certain proteins of interest, we proposed to target the least transcribed regions upstream and/or downstream the gene of interest. To do so, we developed 'universal' template plasmids for donor DNA cassettes with or without a tag, where LoxP sequences may be located either immediately upstream the ATG and downstream the stop codon of the gene of interest, or in the least transcribed areas of intergenic regions. Our methodology is fast, PCR based (molecular cloning-free), highly efficient, versatile, and able to overcome the problems posed by genomic plasticity in Leishmania.

The plaque assay has been widely used for titration of adenovirus (AdV). However, it takes usually 2±3 weeks, so this slow assay often impedes bioprocess development of large-scale AdV production. In this study, we developed a rapid AdV titration assay that can be done within a day. Further, unlike the plaque assay, this assay does not require a laborious serial dilution of samples. This rapid assay can be achieved by using green¯uorescent protein (GFP) as a marker gene and¯ow cytometric analysis. It yields a good correlation between infectious titer of AdV harboring GFP and¯ow cytometric parameters such as average green¯uorescence intensity or % of infected cells. Taken together, this rapid assay will facilitate bioprocess development for ef®cient large-scale AdV production.

Recently, we identified ELL2 as a susceptibility gene for multiple myeloma (MM). To understand its mechanism of action, we performed expression quantitative trait locus analysis in CD138 plasma cells from 1630 MM patients from four populations. We show that the MM risk allele lowers ELL2 expression in these cells (P = 2.5 × 10; β = -0.24 SD), but not in peripheral blood or other tissues. Consistent with this, several variants representing the MM risk allele map to regulatory genomic regions, and three yield reduced transcriptional activity in plasmocytoma cell lines. One of these (rs3777189-C) co-locates with the best-supported lead variants for ELL2 expression and MM risk, and reduces binding of MAFF/G/K family transcription factors. Moreover, further analysis reveals that the MM risk allele associates with upregulation of gene sets related to ribosome biogenesis, and knockout/knockdown and rescue experiments in plasmocytoma cell lines support a cause-effect relationship. Our resul...

Traditional veterinary virus vaccines, such as inactivated and live-attenuated vaccines, have achieved tremendous success in controlling many viral diseases of livestock and chickens worldwide. However, many recent viral outbreaks caused by different emerging and re-emerging viruses continue to be reported annually worldwide. It is therefore necessary to develop new control regimens. Nanoparticle research has received considerable attention in the last two decades as a promising platform with significant success in veterinary medicine, replacing traditional viral vector vaccines. However, the field of nanoparticle applications is still in its initial phase of growth. Here, we discuss various preparation methods, characteristics, physical properties, antiviral effects, and pharmacokinetics of well-developed nanoparticles and the potential of nanoparticles or nano-vaccines as a promising antiviral platform for veterinary medicine.

Background: The papaya mosaic virus (PapMV) vaccine platform is a rod-shape nanoparticle made of the recombinant PapMV coat protein (CP) self-assembled around a non-coding ssRNA template. The PapMV nanoparticle induces innate immunity through stimulation of the toll-like receptors (TLR) 7 and 8. The display of the vaccine antigen at the surface of the nanoparticle, associated with the co-stimulation signal via TLR7/8, ensures a strong stimulation of the immune response, which is ideal for the development of candidate vaccines. In this study, we assess the impact of where the peptide antigen is fused, either at the surface or at the extremities of the nanoparticles, on the immune response directed to that antigen. Methods: Two different peptides from influenza A virus were used as model antigens. The conserved M2e peptide was chosen as the B-cell epitope, and a peptide derived from the nucleocapsid was chosen as the CTL epitope. These peptides were coupled at two different positions ...

Capsid-like particles (CLPs) are multimeric, repetitive assemblies of recombinant viral capsid proteins, which are highly immunogenic due to their structural similarity to wild-type viruses. CLPs can be used as molecular scaffolds to enable the presentation of soluble vaccine antigens in a similar structural format, which can significantly increase the immunogenicity of the antigen. CLP-based antigen display can be obtained by various genetic and modular conjugation methods. However, these vary in their versatility as well as efficiency in achieving an immunogenic antigen display. Here, we make a comparative review of the major CLP-based antigen display technologies. The Tag/Catcher-AP205 platform is highlighted as a particularly versatile and efficient technology that offers new qualitative and practical advantages in designing modular CLP vaccines. Finally, we discuss how split-protein Tag/Catcher conjugation systems can help to further propagate and enhance modular CLP vaccine de...

In this consensus paper resulting from a meeting that involved representatives from more than 20 European partners, we recommend the foundation of an expert group (European Steering Committee) to assess the potential benefits and draw-backs of genome editing (off-targets, mosaicisms, etc.), and to design risk matrices and scenarios for a responsible use of this promising technology. In addition, this European steering committee will contribute in promoting an open debate on societal aspects prior to a translation into national and international legislation.

Duck-targeted vaccines to protect against avian influenza are critically needed to aid in influenza disease control efforts in regions where ducks are endemic for highly pathogenic avian influenza (HPAI). Duck enteritis virus (DEV) is a promising candidate viral vector for development of vaccines targeting ducks, owing to its large genome and narrow host range. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a versatile gene-editing tool that has proven beneficial for gene modification and construction of recombinant DNA viral vectored vaccines. Currently, there are two commonly used methods for gene insertion: non-homologous end-joining (NHEJ) and homology-directed repair (HDR). Owing to its advantages in efficiency and independence from molecular requirements of the homologous arms, we utilized NHEJ-dependent CRISPR/Cas9 to insert the influenza hemagglutinin (HA) antigen expression cassette into the DEV genome. The insert was initially tagged with r...

Citation: Eman Soliman., et al. “Comparative Study Between the Immune Response of Different RHDV Vaccines Used in Rabbit Farms in Egypt”. EC Veterinary Science 5.12 (2020): 84-91. Abstract Eman Soliman1*, Desoky Mohamed Mourad2, Hanan Saad EL Samahy2, Mossad W G3, Fatma EL Zahraa Gamal EL Din1 and Selim Selim Salama1 1Central Laboratory for Evaluation of Veterinary Biologics, Agriculture research center 2Desert Research Center, Agriculture research center 3Veterinary Serum and Vaccine Research Institute, Agriculture research center

Traditional veterinary virus vaccines, such as inactivated and live-attenuated vaccines, have achieved tremendous success in controlling many viral diseases of livestock and chickens worldwide. However, many recent viral outbreaks caused by different emerging and re-emerging viruses continue to be reported annually worldwide. It is therefore necessary to develop new control regimens. Nanoparticle research has received considerable attention in the last two decades as a promising platform with significant success in veterinary medicine, replacing traditional viral vector vaccines. However, the field of nanoparticle applications is still in its initial phase of growth. Here, we discuss various preparation methods, characteristics, physical properties, antiviral effects, and pharmacokinetics of well-developed nanoparticles and the potential of nanoparticles or nano-vaccines as a promising antiviral platform for veterinary medicine.