Human and bovine respiratory syncytial virus vaccine research and development

Human (HRSV) and bovine (BRSV) respiratory syncytial viruses (RSV) are two closely related viruses, which are the most important causative agents of respiratory tract infections of young children and calves, respectively. BRSV vaccines have been available for nearly 2 decades. They probably have reduced the prevalence of RSV infection but their efficacy needs improvement. In contrast, despite decades of research, there is no currently licensed vaccine for the prevention of HRSV disease. Development of a HRSV vaccine for infants has been hindered by the lack of a relevant animal model that develops disease, the need to immunize immunologically immature young infants, the difficulty for live vaccines to find the right balance between attenuation and immunogenicity, and the risk of vaccine-associated disease. During the past 15 years, intensive research into a HRSV vaccine has yielded vaccine candidates, which have been evaluated in animal models and, for some of them, in clinical trials in humans. Recent formulations have focused on subunit vaccines with specific CD4+ Th-1 immune response-activating adjuvants and on genetically engineered live attenuated vaccines. It is likely that different HRSV vaccines and/or combinations of vaccines used sequentially will be needed for the various populations at risk. This review discusses the recent advances in RSV vaccine development

Detection of minority variants within bovine respiratory syncytial virus populations using oligonucleotide-based microarrays

Microarray technology, originally developed for highly parallel examination of gene expression is regarded as a potential tool in prognosis and diagnosis. With respect to a discrimination analysis, difference as small as one nucleotide base can be distinguished using oligonucleotide-basedmicroarrays. However, this degree of specificity is dependent on several parameters, including the size of the oligoprobes and the sequence context of the probes (e.g. local melting temperature), hybridization conditions and to some extent the chemistry of the glass slides onto which the probes are deposited. Using bovine respiratory syncytial virus (BRSV) as a model study, an oligonucleotide-based microarray approach was developed to measure the relative abundance of a particular single nucleotide variant within mixed BRSV populations. Using this technology, we show that it is possible to discriminate at a rate of 1%, minority variants in a BRSV population

In vivo evidence for quasispecies distributions in the bovine respiratory syncytial virus genome

We analyzed the genetic evolution of bovine respiratory syncytial virus (BRSV) isolate W2-00131, from its isolation in bovine turbinate (BT) cells to its inoculation in calves. Results showed that the BRSV genomic region encoding the highly variable glycoprotein G remains genetically stable after virus isolation and over 10 serial infections in BT cells, as well as following experimental inoculation in calves. This remarkable genetic stability led us to examine the mutant spectrum of several populations derived from this field isolate. Sequence analysis of molecular clones revealed an important genetic heterogeneity in G coding region of each population, with mutation frequencies ranging from 6.8 to 10.1 10-4 substitutions/nucleotide. The non-synonymous mutations of the mutant spectrum mapped preferentially within the two variable antigenic regions of the ectodomain or close to the highly conserved domain. These results suggest that RSV populations may evolve as complex and dynamic mutant swarms, despite apparent genetic stability

A new subunit vaccine based on nucleoprotein nanoparticles confers partial clinical and virological protection in calves against bovine respiratory syncytial virus

Human and bovine respiratory syncytial viruses (HRSV and BRSV) are two closely related, worldwide prevalent viruses that are the leading cause of severe airway disease in children and calves, respectively. Efficacy of commercial bovine vaccines needs improvement and no human vaccine is licensed yet. We reported that nasal vaccination with the HRSV nucleoprotein produced as recombinant ringshaped nanoparticles (NSRS) protects mice against a viral challenge with HRSV. The aim of this work was to evaluate this new vaccine that uses a conserved viral antigen, in calves, natural hosts for BRSV. Calves, free of colostral or natural anti-BRSV antibodies, were vaccinated with NSRS either intramuscularly, or both intramuscularly and intranasally using MontanideTM ISA71 and IMS4132 as adjuvants and challenged with BRSV. All vaccinated calves developed anti-N antibodies in blood and nasal secretions and N-specific cellular immunity in local lymph nodes. Clinical monitoring post-challenge demonstrated moderate respiratory pathology with local lung tissue consolidations for the non vaccinated calves that were significantly reduced in the vaccinated calves. Vaccinated calves had lower viral loads than the nonvaccinated control calves. Thus NSRS vaccination in calves provided cross-protective immunity against BRSV infection without adverse inflammatory reaction

S. aureus and E. coli induce IL-32 gene expression to different extents during infection of bovine mammary gland epithelial cells

Introduction: While the role of many cytokines during infection is known, the implication of recently discovered cytokines is not well defined. The involvement of recently describedinterleukin-32 in infectious mastitis, caused by two prevalent mastitis pathogens, Staphylococcus aureus and Escherichia coli, has not been investigated so far and we present initialresults.Material and Methods: Two bovine mammary gland epithelial cells lines (BME-UV and PS) were infected with bovine infectious mastitis organisms E. coli or S. aureus. Using RT- qPCRthe kinetics of IL-32, IL-6 and IL-8 gene expression was determined.Results: E. coli induces a weak IL-6 expression in BME-UV and a strong IL-6 expression in PS cells, while a faint IL-6 expression was observed during S. aureus infection in both celllines. IL-8 expression was detected during the exposure to both pathogens with the strongest signal in E. coli-infected PS cells. Regarding IL-32 expression, different responses wereobserved in cells exposed to E. coli and S. aureus strains: while IL-32 expression was increased in both cell lines infected with E. coli, there was a decrease of IL-32 expression in cellsexposed to S. aureus strains.Conclusion: Taking into account that endogenous IL-32 controls the production of IL-6, IFN-γ and TNF-α during some infection, the decrease of IL-32 expression in cells exposed to S.aureus, could be partially responsible for the weak immune response during staphylococcal infection, which may be favorable for the development of chronic mastitis

Inflammasomes involvement in Staphylococcus aureus infection of human osteoblast-like cells MG-63

Inflammation is a coordinated immune response to infections or tissue damage. The inflammasome is a multi-protein signaling platform that assembles after recognition of danger signals and/or pathogens by a family of cytosolic receptors called NLRs (nucleotide-binding domain and leucine rich repeats containing receptors) or PYHIN protein family members. Once assembled, inflammasomes initiate signaling by activation of downstream proteases, most notably Caspase-1 and Caspase-11, which then proteolytically mature pro-IL-1β, pro-IL-18, and pro-IL-33, and promote their secretion from the cell. Furthermore, inflammasome activation triggers pyroptosis, an inflammatory form of cell death. Staphylococcus aureus is a highly adaptive and versatile gram-positive bacterium that has major importance to human and animal health. S. aureus can cause life-threatening infections such as bacteremia, pneumonia, meningitis, endocarditis and sepsis. S. aureus are also the predominant cause of bone infections worldwide. Comprehending the mechanisms by which staphylococci interact with and damage bone is critical to the development of new approaches to meet this challenge. While the role of inflammasomes formed in the different types of phagocytes during S. aureus infection was widely investigated, the involvement of inflammasomes in osteoblast cells have not been studied. Objective: To understand the mechanisms of Bone Joint Infection we investigated the involvement of inflammasomes in the model of persistent infection of human osteoblast-like cells.Materials and Methods: CRISPR/Cas9 technology was used to prepare Caspase-1 deficient line of MG-63 cells. Western blot analysis and ELISA were employed for the detection of activated cytokines.Results: An employment of wild type vs Caspase-1 deficient MG-63 osteoblast-like cells allow demonstrating the involvement of inflammasomes during S. aureus infection. Using deletion mutants and complemented S. aureus strains, we determined the role of its most important virulence factors for their capacity to activate Caspase-1 and produce IL-1β.Conclusions: Non-phagocytic osteoblast-like MG-63 cells form inflammasomes in the response to S. aureus infection, however the time of inflammasomes formation was different compared to the professional phagocytes. Many virulence factors induce the inflammasomes assemblage