Search Results (177)

The expanding distribution and geographic range of mosquitoes have potentially contributed to increased flaviviral dissemination and transmission. Despite the growing burden of flaviviral infections, there are no effective antiviral treatments or vaccines, highlighting the need for novel therapeutic targets. Tetraspanins, a superfamily of transmembrane domain glycoproteins involved in cellular organization, signaling, and protein–protein interactions have been recognized as potential mediators of flaviviral infection and transmission. While their roles in vertebrate hosts have been explored, their involvement in flaviviral replication and dissemination within medically important vectors remains poorly understood. In this study, we investigated the role of arthropod tetraspanins in mosquito cells and extracellular vesicles (EVs) derived from cells infected with Zika virus (ZIKV) and dengue virus (serotype 2; DENV2). Among several of the tetraspanins analyzed, only CD151 was significantly upregulated in both mosquito cells and in EVs derived from ZIKV/DENV2-infected cells. RNAi-mediated silencing of CD151 led to a marked reduction in viral burden, suggesting its crucial role in flavivirus replication. Inhibition of EV biogenesis using GW4869 further demonstrated that EV-mediated viral transmission contributes to flavivirus propagation. Additionally, co-immunoprecipitation and immunofluorescence analyses revealed direct interactions between CD151 and ZIKV NS2B and DENV2 capsid proteins. Overall, our findings highlight the functional importance of mosquito CD151 in the replication and transmission of ZIKV and DENV2. This study provides new insights into the molecular mechanisms of flaviviral infection in mosquitoes and suggests that targeting vector tetraspanins may offer a potential approach to controlling mosquito-borne flaviviruses. Full article

Self-amplifying RNA-based (saRNA) technology represents the last frontier in using synthetic RNA in vaccinology. Typically, saRNA consists of positive-strand RNA molecules of viral origin (almost exclusively from alphaviruses) where the sequences of structural proteins are replaced with the open reading frame coding the antigen of interest. For in vivo delivery, they are complexed with lipid nanoparticles (LNPs), just like current COVID-19 vaccines based on synthetic messenger RNA (mRNA). Given their ability to amplify themselves inside the cell, optimal intracellular levels of the immunogenic antigen can be achieved by delivering lower amounts of saRNA molecules compared to mRNA-based vaccines. However, the excessive intracellular accumulation of saRNA may represent a relevant drawback since, as already described in alphavirus-infected cells, the recipient cell may react by incorporating excessive RNA molecules into extracellular vesicles (EVs). These EVs can shed and enter neighboring as well as distant cells, where the EV-associated saRNA can start a new replication cycle. This mechanism could lead to an unwanted and unnecessary spread of saRNA throughout the body, posing relevant safety issues. This perspective article discusses the molecular mechanisms through which saRNAs can be transmitted among different cells/tissues. In addition, a simple way to control the possible excessive saRNA intercellular propagation through the co-expression of an EV-anchored protein inhibiting the saRNA replication is proposed. Based on current knowledge, a safety improvement of saRNA-based vaccines appears to be mandatory for their usage in healthy humans. Full article

Extracellular vesicles (EVs) are nanometer-sized lipid structures actively secreted by Gram-negative and Gram-positive bacteria, representing a sophisticated microbial adaptation and communication strategy. These structures are involved in biomolecular transport, the regulation of biological processes, the modulation of host–pathogen interactions, and adaptation to hostile environmental conditions. EVs also play a crucial role in virulence, antibiotic resistance, and biofilm formation. This review will explore the biogenesis, composition, and biological mechanisms of outer membrane vesicles (OMVs) secreted by Gram-negative bacteria and membrane vesicles (MVs) generated by Gram-positive bacteria. In detail, the modulation of EVs in response to antibiotic exposure will be addressed. The role of EV morpho-functional adaptations will be studied in antimicrobial resistance, the gene determinant spread, and survival in adverse environments. This study aims to provide a comprehensive overview of the EV role in bacterial physiology, highlighting their ecological, evolutionary, and biotechnological implications. An overview of the enzymes and proteins mainly involved in OMV-mediated resistance mechanisms will also be provided. These insights could open new perspectives for developing therapeutic strategies that counteract EV secretion and biotechnological applications, such as vaccines and drug delivery systems. Full article

Background: The three commercial Enterovirus 71 (EV71) inactivated vaccines which have effectively controlled the EV71 pandemic currently rely on inherent variable in vivo potency methods for batch release. To align with 3R (Replacement, Reduction, Refinement) principles and enhance quality control, this study referred to WHO guidelines and the European Pharmacopoeia to develop in vitro relative potency (IVRP) methods. Methods: Working standards tracing to phase 3 clinical vaccines were established. Manufacture-specific IVRP methods were developed and validated per ICH Q14/Q2(R2), utilizing conformational epitope-targeting neutralizing monoclonal antibodies (MAbs). One of the MAbs (CT11F9) recognition sites was clarified with Cryo-EM. Subsequently, the performance of IVRP was assessed using varied concentrations and heat-treated vaccines. The correlation between IVRP and in vivo methods was analyzed, followed by setting IVRP specifications. Results: The manufacturer-specific working standard exhibited ED50 values comparable to those of related phase 3 clinical vaccines. All IVRP methods achieved a relative bias/precision/total error ≤ 15%. The IVRP methods correlated with in vivo methods (p < 0.05, r > 0.9) can discriminate EV71 antigen concentrations (p < 0.01, r > 0.99) and indicate the stability of the vaccines. Cryo-EM was adopted to identify the epitopes recognized by CT11F9, revealing that this neutralizing antibody recognizes a conformational epitope spanning VP1-3 of the same protomer. Using 31–47 batches of commercial vaccines, IVRP specifications were proposed as 0.56–1.35, 0.58–1.40, and 0.54–1.50. Conclusions: Based on conformational epitope-targeting neutralizing MAbs, manufacturer-specific IVRP methods, which were sensitive to process variations and correlated with in vivo results, have been established. IVRP methods provide a reliable, animal-free alternative for EV71 vaccine batch release. Full article

Background/Objective: Anti-tick vaccines represent a promising alternative to chemical acaricides for the management of ticks on wildlife; however, little progress has been made to produce a vaccine effective in wild hosts that are critical for tick reproduction, such as the white-tailed deer (Odocoileus virginianus). We recently tested Amblyomma americanum salivary and midgut extracellular vesicles as vaccine candidates in white-tailed deer, which resulted in on-host female tick mortality. The objective of this study was to identify the proteins recognized by the antibodies regenerated during these vaccinations to determine potential antigens for vaccine development for white-tailed deer. Methods: Using a proteomic approach, we characterized the cargo within salivary and midgut vesicles. Label-free quantitative proteomics were used to investigate significant changes in protein loading within extracellular vesicles in these two organs. The pre-vaccination and post-vaccination serum from three animals vaccinated with salivary and midgut vesicles and one control animal were used to identify proteins recognized by circulating antibodies. Results: We show that these salivary and midgut vesicles contain a “core-cargo” enriched in chaperones, small GTPases, and other proteins previously reported in small EVs. Label-free quantitative proteomics show significant differences in protein cargo between salivary and midgut vesicles (333 proteins out of 516). Proteomic analysis of immunoprecipitated proteins identified thirty antigens with potential for use in anti-tick vaccines, seven of which we have categorized as high priority. Conclusions: Proteins within tick salivary and midgut vesicles are recognized by antibodies from vaccinated white-tailed deer. These proteins can be further evaluated for their function and potential as vaccine candidates against ticks. Full article

Background/Objectives: Enterovirus 71 (EV71) and coxsackieviruses A16 (CA16), A10 (CA10), and A6 (CA6) are the primary pathogens that cause hand, foot, and mouth disease (HFMD). Currently, many manufacturers are developing bivalent, trivalent, and tetravalent vaccines that target these antigens. Cell-based neutralization assay (CBNA), the gold standard for detecting neutralizing antibodies (NtAbs), which are used as indicators of HFMD vaccine efficacy, has several limitations. We aimed to develop a novel assay for detecting NtAbs against a quadrivalent HFMD vaccine. Methods: We developed a four-color pseudovirus-based neutralization assay (PBNA), utilizing fluorescent reporter genes, to rapidly evaluate neutralizing antibodies against EV71, CA16, CA10, and CA6 in multivalent vaccines and compared it with CBNA. Results: PBNA could rapidly and simultaneously detect NtAbs against the four serotypes and required lesser amounts of sera compared to CBNA. A good consistency in determining NtAb titers was observed for PBNA and CBNA. Conclusions: PBNA provides a robust tool for evaluating the efficacy of multivalent HFMD vaccines and conducting seroepidemiological studies. Full article

Hand, Foot, and Mouth Disease (HFMD) is a viral illness caused by enterovirus infections. While the introduction of the enterovirus 71 (EV71) vaccine has significantly reduced the number of EV71-related cases, the continued spread of Coxsackievirus A16 (CVA16) remains a major public health threat. Previous studies have shown that human SCARB2 (hSCARB2) knock-in (KI) mice, generated using embryonic stem cell (ESC) technology, are susceptible to CVA16. However, these models have failed to reproduce the clinical pathology and neurotoxicity after CVA16 infection. Therefore, there is an urgent need for a more reliable and effective animal model to study CVA16. In this study, we successfully created a hSCARB2 KI mouse model targeting the ROSA26 locus using CRISPR/Cas9 gene editing technology. The application of CRISPR/Cas9 enabled stable and widespread expression of hSCARB2 in the model. After infection, the KI mice exhibited a clinical pathology that closely mimics human infection, with prominent limb weakness and paralysis. The virus was detectable in multiple major organs of the mice, with peak viral load observed on day 7 post-infection, gradually clearing thereafter. Further analysis revealed widespread neuronal necrosis and infiltration of inflammatory cells in the brain and spinal cord of the KI mice. Additionally, significant activation of astrocytes (GFAP-positive) and microglia (IBA1-positive) was observed in the brain, suggesting that CVA16 infection may induce limb paralysis by attacking neuronal cells. Overall, this model effectively replicates the neuropathological changes induced by CVA16 infection and provides a potential experimental platform for studying CVA16-associated pathogenesis and neurotoxicity. Full article

Cell-derived, nanoscale extracellular vesicles (EVs) have emerged as promising tools in diagnostic, therapeutic, and vaccine applications. Their unique properties including the capability to encapsulate diverse molecular cargo as well as the versatility in surface functionalization make them ideal candidates for safe and effective vehicles to deliver a range of biomolecules including gene editing cassettes, therapeutic proteins, glycans, and glycoconjugate vaccines. In this review, we discuss recent advances in the development of EVs derived from mammalian and bacterial cells for use in a delivery of carbohydrate-based protein therapeutics and vaccines. We highlight key innovations in EVs’ molecular design, characterization, and deployment for treating diseases including Alzheimer’s disease, infectious diseases, and cancers. We discuss challenges for their clinical translation and provide perspectives for future development of EVs within biopharmaceutical research and the clinical translation landscape. Full article

The human papillomavirus (HPV) has been associated with the carcinogenesis of cutaneous squamous cell carcinoma (cSCC), especially in immunosuppressed patients. This article reviews the microbiology of HPV and its role in tissue tropism, invasion, and oncogenesis. It also describes possible HPV oncogenic ability due to the inactivation of the host p53 and retinoblastoma protein (pRb) by HPV oncoproteins E6 and E7, producing a suppression of cell cycle checkpoints and uncontrolled cell proliferation that may eventually result in invasive carcinoma. We will focus on β-HPV types and their role in epidermodysplasia verruciformis (EV), as well as α types and their ability to cause cutaneous and mucosal pathology. We also intend to examine the clinical characteristics of cSCC related to HPV and host immunosuppression conditions such as solid organ transplant in order to provide management guidelines for patients with cSCC associated with HPV based on available data. Other topics addressed in this article include particular locations of cSCC, such as nails; the prognosis; the recurrence; therapeutic modalities; and the role of HPV vaccines. Full article

Antimicrobial resistance (AMR) is a growing global health crisis that threatens the efficacy of antibiotics and modern medical interventions. The emergence of multidrug-resistant (MDR) pathogens, exacerbated by the misuse of antibiotics in healthcare and agriculture, underscores the urgent need for innovative solutions. (1) Background: AMR arises from complex interactions between human, animal, and environmental health, further aggravated by the overuse and inadequate regulation of antibiotics. Conventional treatments are increasingly ineffective, necessitating alternative strategies. Emerging approaches, including bacteriophage therapy, antimicrobial peptides (AMPs), nanotechnology, microbial extracellular vesicles (EVs), and CRISPR-based antimicrobials, provide novel mechanisms that complement traditional antibiotics in combating resistant pathogens. (2) Methods: This review critically analyzes advanced antibacterial strategies in conjunction with systemic reforms such as antimicrobial stewardship programs, the One Health framework, and advanced surveillance tools. These methods can enhance resistance detection, guide interventions, and promote sustainable practices. Additionally, economic, logistical, and regulatory challenges impeding their implementation are evaluated. (3) Results: Emerging technologies, such as CRISPR and nanotechnology, exhibit promising potential in targeting resistance mechanisms. However, disparities in resource distribution and regulatory barriers hinder widespread adoption. Public–private partnerships and sustainable agriculture practices are critical to overcoming these obstacles. (4) Conclusions: A holistic and integrated approach is essential for mitigating the impact of AMR. By aligning innovative therapeutic strategies with global health policies, fostering interdisciplinary collaboration, and ensuring equitable resource distribution, we can develop a sustainable response to this 21st-century challenge. Full article

Background: In 2024, mainland China witnessed a significant upsurge in Hand, Foot, and Mouth Disease (HFMD) cases. Coxsackievirus A16 (CVA16) is one of the primary causative agents of HFMD. Long-term monitoring of theCVA16 infection rate and genotype changes is crucial for the prevention and control of HFMD. Methods: A total of 40,673 clinical specimens were collected from suspected HFMD cases in Guangdong province from 2018 to 2024, including rectal swabs (n = 27,954), throat swabs (n = 6791), stool (n = 5923), cerebrospinal fluid (n = 3), and herpes fluid (n = 2). A total of 24,410 samples were detected as EV-positive and further typed by RT-PCR. A total of 872 CVA16-positive samples were isolated and further sequenced to obtain the full-length VP1 sequence. Phylogenetic analysis was performed based on viral protein 1 gene (VP1). Results: In the first 25 weeks of 2024, reported cases of HFMD were 1.36 times higher than the mean rates of 2023. In 2024, CVA16 predominated at 75.42%, contrasting with the past etiological pattern in which the CVA6 was predominant with the detection rate ranging from 32.85 to 77.61% from 2019 to 2023. Phylogenetic analysis based on the VP1 gene revealed that the B1a and B1b subtypes co-circulated in Guangdong from 2018 to 2022. The B1c outbreak clade, detected in Guangdong in 2023, constituted 68.24% of the 148 strains of CVA16 collected in 2024, suggesting a subtype shift in the CVA16 virus. There were three specific amino acid variations (P3S, I235V, and T240A) in the VP1 sequence of B1c. Conclusions: The new emergence of the CVA16 B1c outbreak clade in Guangdong during 2023–2024 highlights the necessity for the enhanced surveillance of the virus evolution epidemiological dynamic in this region. Furthermore, it is imperative to closely monitor the etiological pattern changes in Hand, Foot, and Mouth Disease (HFMD) in other regions as well. Such vigilance will be instrumental in guiding future vaccination strategies for HFMD. Full article

Hand, foot, and mouth disease (HFMD) represents an emerging health concern whose main causative agents are Coxsackievirus A6 (CVA6) and enterovirus A71 (EV71). The lack of a CVA6 vaccine and the rise of new HFMD-causing strains due to the containment of established HFMD-causing viruses necessitates the search for alternative vaccine technologies, including virus-like particle (VLP) vaccine candidates. While studies have demonstrated that production of enterovirus-like particles in various organisms can be achieved by expression of the viral P1 structural proteins and the 3CD protease, optimization based on the interplay between the three most commonly altered infection parameters (multiplicity of infection (MOI), viable cell density at the time of infection (VCD), and the infection period) is often not investigated. To address this challenge, we have performed Design of Experiments (DoE) to optimize the production of CVA6 and EV71 VLPs. Our results indicate that CVA6 VLP production peaks at high MOI, high VCD, and long infection periods. Our subsequent downstream purification processes yielded 38 mg and 158 mg of purified CVA6 and EV71 VLPs from 1 L crude harvest, respectively. This translates into thousands of potential vaccine doses and highlights the economic potential of enterovirus-like particles for vaccine purposes. Full article

Human enterovirus 71 (EV-A71), a member of the Picornaviridae family, is predominantly associated with hand, foot, and mouth disease in infants and young children. Additionally, EV-A71 can cause severe neurological complications, including aseptic meningitis, brainstem encephalitis, and fatalities. The molecular mechanisms underlying these symptoms are complex and involve the viral tissue tropism, evasion from the host immune responses, induction of the programmed cell death, and cytokine storms. This review article delves into the EV-A71 life cycle, with a particular emphasis on recent advancements in understanding the virion structure, tissue tropism, and the interplay between the virus and host regulatory networks during replication. The comprehensive review is expected to contribute to our understanding of EV-A71 pathogenesis and inform the development of antiviral therapies and vaccines. Full article

Enterovirus 68 (EV-D68) is a non-enveloped virus with a positive-sense single-stranded RNA genome that causes respiratory diseases and acute flaccid myelitis, posing significant threats to human health. However, an effective vaccine remains undeveloped. SIRT1, a nicotinamide adenine dinucleotide (NAD+)-dependent enzyme, plays a key role in cellular metabolism, but its interaction with NAD+ during viral infections is not well understood. In this study, through a metabolomics analysis, we demonstrate that EV-D68 infection influences cellular metabolism. Additionally, we show that NAD+ inhibits EV-D68 infection both in vivo and in vitro. EV-D68 reduces cellular NAD+ levels by regulating the expression of enzymes involved in NAD+ consumption and synthesis. Moreover, the infection increases the expression of sirtuin 1 (SIRT1), which inhibits EV-D68 replication in turn. Mechanistically, SIRT1 suppresses EV-D68 5′UTR-mediated translation, and the antiviral effect of SIRT1 on EV-D68 replication is enhanced by NAD+. Collectively, our findings highlight the critical role of NAD+ metabolism in EV-D68 infection and reveal the antiviral potential of SIRT1, providing valuable insights for the development of antiviral strategies. Full article

This study aimed to create a new recombinant virus by modifying the EV-A71 capsid protein, serving as a useful tool and model for studying human Enteroviruses. We developed a new screening method using EV-A71 pseudovirus particles to systematically identify suitable insertion sites and tag types in the VP1 capsid protein. The pseudovirus’s infectivity and replication can be assessed by measuring postinfection luciferase signals. We reported that the site after the 100th amino acid within the VP1 BC loop of EV-A71 is particularly permissive for the insertion of various tags. Notably, the introduction of S and V5 tags at this position had minimal effect on the fitness of the tagged pseudovirus. Furthermore, recombinant infectious EV-A71 strains tagged with S and V5 epitopes were successfully rescued, and the stability of these tags was verified. Computational analysis suggested that viable insertions should be compatible with capsid assembly and receptor binding, whereas non-viable insertions could potentially disrupt the capsid’s binding with heparan sulfate. We expect the tagged recombinant EV-A71 to be a useful tool for studying the various stages of the enterovirus life cycle and for virus purification, immunoprecipitation, and research in immunology and vaccine development. Furthermore, this study serves as a proof of principle and may help develop similar tags in enteroviruses, for which there are fewer available tools. Full article