Applications of the hollow-fibre infection model (HFIM) in viral infection studies

Conventional cell culture systems involve growing cells in stationary cultures in the presence of growth medium containing various types of supplements. At confluency, the cells are divided and further expanded in new culture dishes. This passage from confluent monolayer to sparse cultures does not reflect normal physiological conditions and represents quite a drastic physiological change that may affect the natural cell physiobiology. Hollow-fibre bioreactors were in part developed to overcome these limitations and since their inception, they have widely been used in production of monoclonal antibodies and recombinant proteins. These bioreactors are increasingly used to study antibacterial drug effects via simulation of in vivo pharmacokinetic profiles. The use of the hollow-fibre infection model (HFIM) in viral infection studies is less well developed and in this review we have analysed and summarized the current available literature on the use of these bioreactors, with an emphasis on viruses. Our work has demonstrated that this system can be applied for viral expansion, studies of drug resistance mechanisms, and studies of pharmacokinetic/pharmacodynamic (PK/PD) of antiviral compounds. These platforms could therefore have great applications in large-scale vaccine development, and in studies of mechanisms driving antiviral resistance, since the HFIM could recapitulate the same resistance mechanisms and mutations observed in vivo in clinic. Furthermore, some dosage and spacing regimens evaluated in the HFIM system, as allowing maximal viral suppression, are in line with clinical practice and highlight this 'in vivo-like' system as a powerful tool for experimental validation of in vitro-predicted antiviral activities

Longevity and neutralisation activity of secretory IgA following SARS-CoV-2 infection

The mucosal barrier is a primary defence against inhaled pathogens, comprising secretory antibodies which have the potential to block viral entry and neutralise infection. There is an ongoing need for greater understanding of the mucosal immunity to SARS-CoV-2 infection. In this study, we investigated mucosal IgA through non-invasive saliva sampling of healthcare workers. A total of 551 saliva samples were collected from staff at Great Ormond Street Children’s Hospital who previously tested positive for COVID-19. Participant metadata included age, gender, ethnicity and symptoms. IgA titres were measured by ELISA against viral antigens spike protein, nucleocapsid protein, and spike receptor-binding domain. SARS-CoV-2 neutralisation was measured using a VERO E6 cell culture infection assay. We found that approximately 30% of saliva samples contained detectable IgA specific for at least one of the SARS-CoV-2 antigens. IgA levels in saliva decreased with the time post-infection, and were largely undetectable after six months. IgA titres specific to SARS-CoV-2 were lowest in participants over 60 years old. Specific saliva samples were identified which effectively neutralised SARS-CoV-2 virus infection of epithelial cells. Our results suggest secretory IgA specific to SARS-CoV-2 can be detected in saliva following infection, an accessible sample type for testing, although titres decreased over time. Some saliva samples were able to neutralise SARS-CoV-2 infectivity against cultured epithelial cells. This data could be used to assess the risk of re-infection with SARS-CoV-2, as well as accelerate efforts to develop effective mucosal vaccination with longer lasting protection

Tilapia Lake Virus Vaccine Development: A Review on the Recent Advances

Tilapia tilapinevirus (or tilapia lake virus, TiLV) is a recently emerging virus associated with a novel disease affecting and decimating tilapia populations around the world. Since its initial identification, TiLV has been reported in 17 countries, often causing mortalities as high as 90% in the affected populations. To date, no therapeutics or commercial vaccines exist for TiLV disease control. Tilapia exposed to TiLV can develop protective immunity, suggesting that vaccination is achievable. Given the important role of vaccination in fish farming, several vaccine strategies are currently being explored and put forward against TiLV but, a comprehensive overview on the efficacy of these platforms is lacking. We here present these approaches in relation with previously developed fish vaccines and discuss their efficacy, vaccine administration routes, and the various factors that can impact vaccine efficacy. The overall recent advances in TiLV vaccine development show different but promising levels of protection. The field is however hampered by the lack of knowledge of the biology of TiLV, notably the function of its genes. Further research and the incorporation of several approaches including prime–boost vaccine regimens, codon optimization, or reverse vaccinology would be beneficial to increase the effectiveness of vaccines targeting TiLV and are further discussed in this review

Mapping of Tilapia Lake Virus entry pathways with inhibitors reveals dependence on dynamin activity and cholesterol but not endosomal acidification

Tilapia Lake Virus (TiLV) is an emerging virus lethal to tilapia, which threatens the global tilapia aquaculture with severe implications for food security. TiLV possesses similar features to orthomyxoviruses but is classified in the sole and the monotypic genus Tilapinevirus of the family Amnoonviridae. TiLV enveloped virions encapsidate a genome comprising ten segments of single-stranded, negative RNA. Remarkably, nine of TiLV’s ten major proteins lack sequence homology to any known viral or cellular proteins. The mode of TiLV entry into tilapia cells is not known. Following the measurement of the entry window of TiLV (∼3 h), we applied a panel of inhibitors of known regulators of endocytic functions to map the molecular requirements for TiLV entry. We identified productive entry by quantification of TiLV nucleoprotein expression and the generation of infectious particles. Inhibition of dynamin activity with dynasore or dynole, or depletion of cholesterol with methyl-β-cyclodextrin, strongly inhibited TiLV protein synthesis and infectious virion production. Moreover, inhibition of actin cytoskeleton polymerization with latrunculin A or microtubule polymerization with nocodazole within the entry window resulted in partial inhibition of TiLV infection. In contrast, inhibitors of endosomal acidification (NH4Cl, bafilomycin A1, or chloroquine), an inhibitor of clathrin-coated pit assembly (pitstop 2), and erlotinib—an inhibitor of the endocytic Cyclin G-associated kinase (GAK), did not affect TiLV entry. Altogether, these results suggest that TiLV enters via dynamin-mediated endocytosis in a cholesterol-, cytoskeleton-dependent manner, and clathrin-, pH-independent manner. Thus, despite being an orthomyx-like virus, when compared to the prototypical orthomyxovirus (influenza A virus), TiLV shows a distinct set of requirements for entry into cells

Knowns and unknowns of TiLV-associated neuronal disease

Tilapia Lake Virus (TiLV) is associated with pathological changes in the brain of infected fish, but the mechanisms driving the virus's neuropathogenesis remain poorly characterized. TiLV establishes a persistent infection in the brain of infected fish even when the virus is no longer detectable in the peripheral organs, rendering therapeutic interventions and disease management challenging. Moreover, the persistence of the virus in the brain may pose a risk for viral reinfection and spread and contribute to ongoing tissue damage and neuroinflammatory processes. In this review, we explore TiLV-associated neurological disease. We discuss the possible mechanism(s) used by TiLV to enter the central nervous system (CNS) and examine TiLV-induced neuroinflammation and brain immune responses. Lastly, we discuss future research questions and knowledge gaps to be addressed to significantly advance this field

Salivary IgA and vimentin differentiate in vitro SARS-CoV-2 infection: a study of 290 convalescent COVID-19 patients

SARS-CoV-2 initially infects cells in the nasopharynx and oral cavity. The immune system at these mucosal sites plays a crucial role in minimizing viral transmission and infection. To develop new strategies for preventing SARS-CoV-2 infection, this study aimed to identify proteins that protect against viral infection in saliva. We collected 551 saliva samples from 290 healthcare workers who had tested positive for COVID-19, before vaccination, between June and December 2020. The samples were categorized based on their ability to block or enhance infection using in vitro assays. Mass spectrometry and ELISA experiments were used to identify and measure the abundance of proteins that specifically bind to SARS-CoV-2 antigens. IgA specific to SARS-CoV-2 antigens was detectable in over 83% of the convalescent saliva samples. We found that concentrations of anti-RBD IgA >500 pg/µg total protein in saliva correlates with reduced viral infectivity in vitro. However, there is a dissociation between the salivary IgA response to SARS-CoV-2, and systemic IgG titres in convalescent COVID19 patients. Then, using an innovative technique known as spike-baited mass spectrometry, we identified novel spike-binding proteins in saliva, most notably vimentin, which correlated with increased viral infectivity in vitro, could serve as a therapeutic target against COVID-19