[Virus-Like Particles Carrying HIV-1 Env with a Modulated Glycan Composition].

Previously obtained highly immunogenic Env-VLPs ensure overcoming the natural resistance of HIV-1 surface proteins associated with their low level of incorporation and inaccessibility of conserved epitopes to induce neutralizing antibodies. We also adopted this technology to modify Env trimers of the ZM53(T/F) strain to produce Env-VLPs by recombinant vaccinia viruses (rVVs). For VLP production, rVVs expressing Env, Gag-Pol (HIV-1/SIV), and the cowpox virus hr gene, which overcomes the restriction of vaccinia virus replication in CHO cells, were used.

CMT2 and distal hereditary motor neuropathy associated with VRK1 variants: Case series.

Axonal Charcot-Marie-Tooth disease (CMT2) and distal hereditary motor neuropathy (dHMN) are associated with a heterogeneous group of genes encoding proteins that are involved in axonal transport, control of RNA metabolism, mitochondrial dynamics and DNA repair. VRK1 (vaccinia-related kinase 1) is a serine/threonine kinase which is widely expressed in human tissue and plays a role in RNA maturation and processing and in DNA damage response. Variants of VRK1 have been associated with neurodevelopmental and neuromuscular disorders including pontocerebellar hypoplasia, motor neuron disorders and distal hereditary motor neuropathy.

Immunogenic recombinant Mayaro virus-like particles present natively assembled glycoprotein.

Virus-like particles (VLPs) are an established vaccine platform and can be strong immunogens capable of eliciting both humoral and cellular immune responses against a range of pathogens. Here, we show by cryo-electron microscopy that VLPs of Mayaro virus, which contain envelope glycoproteins E1-E2 and capsid, exhibit an architecture that closely resembles native virus. In contrast to monomeric and soluble envelope 2 (E2) glycoprotein, both VLPs as well as the adenovirus and modified vaccinia virus Ankara (MVA) vaccine platforms expressing the equivalent envelope glycoproteins E1-E2, and capsid induced highly neutralising antibodies after immunisation.

Concurrent outbreaks of mpox in Africa-an update.

In this Review, we examine the concurrent outbreaks of mpox in Africa, focusing on clade 1a, the newly emerged clade 1b, and clade 2b lineage A, and how they differ from the 2022 global outbreak caused by clade 2b lineage B.1. Historically, clades 1a and 2a have caused sporadic, small outbreaks in central and west Africa, respectively, primarily through zoonotic transmission.

Plaque reduction neutralization test for smallpox vaccines: Laboratory optimization and validation method for immunogenicity assessment.

The eradication of smallpox, a historic triumph in global public health, was accomplished without a complete conception of the mechanisms underlying vaccine-induced protection. Contemporary concerns regarding potential bioterrorism threats and the possibility of smallpox reemergence have spurred research efforts toward developing third-generation vaccines capable of effectively neutralizing the variola virus. Clinical trials for a third-generation smallpox vaccine (KVAC103) are underway to obtain licensure.

Recombinant Vaccine Production: Production of a Recombinant CCHF MVA Vaccine.

One of the key interventions against infection is immunization, including an increasing focus on development of vaccines against pathogenic bunyaviruses. Whilst different vaccine development approaches exist, recombinant viral vaccines have a strong safety record, are rapid to produce, are cost-effective, and have been demonstrated to be rolled out in response to outbreaks, including in low- and middle-income countries. One viral vector, modified Vaccinia Ankara (MVA), has been used to develop vaccine candidates against Crimean-Congo Haemorrhagic Fever (CCHF) virus through incorporation of the nucleoprotein (NP) and glycoprotein (GP) regions, with the former candidate having now progressed to being the first vaccine against CCHF virus to enter Phase 1 clinical trials.

A multi-antigen vaccinia vaccine broadly protected mice against SARS-CoV-2 and influenza A virus while also targeting SARS-CoV-1 and MERS-CoV.

Introduction: Coronaviruses and influenza viruses are significant respiratory pathogens that cause severe disease burdens and economic losses for society. Due to their diversity and evolution, vaccines typically require periodic updating to remain effective. An additional challenge is imposed by the possible coinfection of SARS-CoV-2 and influenza, which could increase disease severity.

A comparative exploration of mRNA capping enzymes.

With the wide application of messenger RNA (mRNA) technology in medicine and vaccine fields, higher requirements are put forward for mRNA expression efficiency . Since the 5' cap structure can spatially protect mRNA from exonuclease degradation and enhance the initiation of translation reactions, mRNA caps are a promising option to improve the efficiency of mRNA expression . In order to obtain more efficient mRNA capping enzymes, seven mRNA capping enzymes from different viral sources were explored in this study.

Generating MVA-Vector Vaccine Candidates and Testing Them in Animal Models.

Modified Vaccinia Virus Ankara (MVA) is a highly attenuated and replication-deficient vaccinia virus developed through serial passages in chicken embryo fibroblasts (CEF). MVA is increasingly used in biomedicine for vaccine development in preclinical and clinical studies in humans. Major benefits of MVA include a well-established record in clinical safety, long-standing experience in genetic engineering of the virus, a large data set demonstrating efficacy in preclinical models with the capacity to induce both protective antigen-specific antibody and cellular immune responses, and the availability of virus production under Good Manufacturing Practice (GMP) suitable for industrial scale amplification.

Modified Vaccinia Virus Ankara Titration Using Crystal Violet- or Immuno-Staining in DF-1 Cells.

Modified Vaccinia virus Ankara (MVA) is a promising vaccine vector with an outstanding safety profile. The gold standard method to titrate MVA is to immune stain plaques formed in MVA-infected monolayer of primary chicken embryo fibroblasts. Recently, DF-1, an immortal chicken embryo fibroblast line, has also been used.

Sample Preparation for Global Metabolic Profiling of Vaccinia Virus-Infected Primary Human Foreskin Fibroblasts.

Vaccinia virus (VACV), the prototype member of the Poxviridae family, has played a crucial role in medicine as a key component in the development of smallpox vaccines, contributing to the eradication of this deadly disease. Beyond its historical significance, VACV continues to be pivotal in researching metabolic alterations induced by viral infections. Studies have revealed that VACV can impact pathways such as glycolysis, the tricarboxylic acid (TCA) cycle, and lipid metabolism in host cells, offering valuable insights into host-virus interactions and broader cellular metabolism.

Experimental Evolution of Poxviruses.

Experimental evolution is the process of exposing virus populations to defined selective pressures in a laboratory setting to identify adaptive changes. Coupled with deep sequencing, this experimental approach allows for nucleotide-level resolution of poxvirus adaptive strategies over time. Here, we present a general method of poxvirus experimental evolution, Illumina-based deep sequencing, and bioinformatic analyses to identify structural changes (e.

High-Throughput Single-Cell Analysis of Vaccinia Virus Infection.

Here, we describe a robust and flexible method for analyzing the infection of single cells with a fluorescent-reporter virus, with real-time tracking of infected cells through microscopy. We subsequently generate quantitative data from the resulting time-lapse microscopy, and harness these data to generate biologically meaningful parameters at scale. Our methodology offers a practical solution for researchers seeking to understand the complexities and variability of virus-host interactions at a granular level.

Methods to Study Poxvirus Structures by Cryo-EM Imaging Modalities.

Poxviruses are double-stranded DNA viruses that represent the largest known highly pathogenic viruses infecting humans. They undergo dramatic morphological changes during their maturation process, resulting in structural differences between each virion, and their surface is decorated with more than a dozen randomly distributed surface proteins that facilitate viral entry. These are the main reasons poxviruses have eluded high-resolution structure determination.

Evaluation of Vaccinia Virus Infection in Mice Using Two-Reporter Recombinant Virus.

The family Poxviridae comprises multiple viruses with large double-stranded (ds) DNA genomes that can infect numerous vertebrate and invertebrate hosts, including humans. The development of genetic engineering methods for Vaccinia virus (VACV), the prototypic member in the family, have allowed the manipulation of the genomes of poxviruses for the generation of recombinant (r)VACV expressing easily traceable luciferase and/or fluorescent reporter genes. These recombinant viruses have significantly contributed to progress in the field of poxvirus research and accelerated the development of novel prophylactic vaccines and therapeutic antiviral treatments.

Use of a Poxvirus K3 Ortholog as a Positive Selection Marker for Construction of Recombinant Vaccinia Virus with Modified Host Range.

Vaccinia virus (VACV) demonstrates a wide host range, which is determined by its host range genes including the E3L and K3L. The E3L and K3L deletion mutant VACV (VACVΔE3ΔK3) is only able to replicate in cells defective in PKR and RNase L activity. Interestingly, by expressing a K3 ortholog from another poxvirus, the host range of the VACVΔE3ΔK3 can be fine-tuned to specific host species.

Cas9-Mediated Poxvirus Recombinant Recovery (CASPRR) for Fast Recovery of Recombinant Vaccinia Viruses.

Generation of recombinant vaccinia viruses opens many avenues for poxvirus research; however current methods for virus production can be laborious. Traditional methods rely on recombination strategies that produce engineered viruses at a low frequency, which then need to be identified and isolated from a large background of parent virus. For this reason, marker and reporter genes are often included, but in many cases these require removal in subsequent steps and the entire process is relatively inefficient.

Rapid Generation of Recombinant Poxviruses Using CRISPR/Cas9 Gene Editing.

The low-frequency natural recombination that is detected in poxvirus-infected cells has long been used to genetically modify poxviruses. Such recombinant poxviruses have found many applications as vaccines for preventing infectious diseases and as experimental cancer therapeutics. Unfortunately, these methods are time consuming, can leave behind "scars" or selectable markers, and many months of work may be required to generate plaque-purified recombinants bearing multiple virus gene substitutions, deletions, and/or inserted transgenes.