Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to develop protective measures for the human population. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions severely hampers timely characterization of such viruses. We tested heat, formalin, Triton X-100, and ␤-propiolactone treatments for their potencies in inactivating human influenza A(H3N2) and avian A(H7N3) viruses, as well as seasonal and pandemic A(H1N1) virus isolates, while allowing the specimens to retain their virological and immunological properties. Successful heat inactivation coincided with the loss of hemagglutinin (HA) and neuraminidase (NA) characteristics, and ␤-propiolactone inactivation reduced the hemagglutination titer and NA activity of the human influenza virus 10-fold or more. Although Triton X-100 treatment resulted in inconsistent HA activity, the NA activities in culture supernatants were enhanced consistently. Nonetheless, formalin treatment permitted the best retention of HA and NA properties. Triton X-100 treatment proved to be the easiest-to-use influenza virus inactivation protocol for application in combination with phenotypic NA inhibitor susceptibility assays, while formalin treatment preserved B-cell and T-cell epitope antigenicity, allowing the detection of both humoral and cellular immune responses. In conclusion, we demonstrated successful influenza virus characterization using formalin-and Triton X-100-inactivated virus samples. Application of these inactivation protocols limits work under BSL-3 conditions to virus culture, thus enabling more timely determination of public health impact and development of protective measures when a new influenza virus, e.g., pandemic A(H1N1)v virus, is introduced in humans.

The immune system potentially plays an important mechanistic role in the relation between shift work and adverse health effects. To better understand the immunological effects of shift work, we compared numbers and functionality of immune cells between night-shift and non-shift workers. Blood samples were collected from 254 night-shift and 57 non-shift workers employed in hospitals. Absolute numbers of monocytes, granulocytes, lymphocytes, and T cell subsets were assessed. As read out of immune function, monocyte cytokine production and proliferative capacity of CD4 and CD8 T cells in response to various stimuli were analysed. The mean number of monocytes was 1.15 (95%-CI = 1.05–1.26) times higher in night-shift than in non-shift workers. Furthermore, night-shift workers who worked night shifts in the past three days had a higher mean number of lymphocytes (B = 1.12 (95%-CI = 1.01–1.26)), T cells (B = 1.16 (95%-CI = 1.03–1.31)), and CD8 T cells (B = 1.23 (95%-CI = 1.05–1.45)) compared to non-shift workers. No differences in functional parameters of monocytes and lymphocytes were observed. The differences in numbers of monocytes and T cells suggest that chronic exposure to night-shift work as well as recent night-shift work may influence the immune status of healthcare workers. This knowledge could be relevant for preventive initiatives in night-shift workers, such as timing of vaccination.

T cells are essential players in the defense against infection. By targeting the MHC class I antigen-presenting pathway with peptide-based vaccines, antigen-specific T cells can be induced. However, low immunogenicity of peptides poses a challenge. Here, we set out to increase immunogenicity of influenza-specific CD8+ T cell epitopes. By substituting amino acids in wild type sequences with non-proteogenic amino acids, affinity for MHC can be increased, which may ultimately enhance cytotoxic CD8+ T cell responses. Since preventive vaccines against viruses should induce a broad immune response, we used this method to optimize influenza-specific epitopes of varying dominance. For this purpose, HLA-A*0201 epitopes GILGFVFTL, FMYSDFHFI and NMLSTVLGV were selected in order of decreasing MHC-affinity and dominance. For all epitopes, we designed chemically enhanced altered peptide ligands (CPLs) that exhibited greater binding affinity than their WT counterparts; even binding scores of the high affinity GILGFVFTL epitope could be improved. When HLA-A*0201 transgenic mice were vaccinated with selected CPLs, at least 2 out of 4 CPLs of each epitope showed an increase in IFN-γ responses of splenocytes. Moreover, modification of the low affinity epitope NMLSTVLGV led to an increase in the number of mice that responded. By optimizing three additional influenza epitopes specific for HLA-A*0301, we show that this strategy can be extended to other alleles. Thus, enhancing binding affinity of peptides provides a valuable tool to improve the immunogenicity and range of preventive T cell-targeted peptide vaccines.

Background People aged 60 years or older are at high risk for respiratory infections, one of the leading causes of mortality worldwide. Vaccination is the main way to protect against these infections; however, vaccination is less effective in older adults than in younger adults due to ageing of the immune system, so innovative strategies that improve vaccine responses could provide a major public health benefit. The gut microbiota regulates host immune homoeostasis and response against pathogens, but human studies showing the effects of the gut microbiota on respiratory infections in older adults are sparse. We aimed to investigate the composition of the microbiota in relation to respiratory infections and local and systemic immune markers in older adults during an influenza season.Methods In this observational study, participants were selected from an influenza-like illness (ILI) prospective surveillance cohort in which community-dwelling adults aged 60 years and older in the Netherlands were recruited through their general practitioner or the Civil Registry. Inclusion criteria have been described elsewhere. Participants completed questionnaires and self-reported symptoms. To measure microbiota composition, faecal samples were collected from participants registering an ILI event, with a follow-up (recovery) sample collected 7-9 weeks after the ILI event, and from asymptomatic participants not reporting any event throughout the season. We tested associations between microbiota profiles and a set of health-related variables, patient characteristics, and local and systemic immune markers. We cultured identified bacterial biomarkers for ILI with CaCo-2 cells in an in vitro intestinal epithelial model and measured the induced immune response. This study is registered with http://www.trialregister.nl, NL4666.

Currently licensed influenza vaccines mainly induce antibodies against highly variable epitopes. Due to antigenic drift, protection is subtype or strain-specific and regular vaccine updates are required. In case of antigenic shifts, which have caused several pandemics in the past, completely new vaccines need to be developed. We set out to develop a vaccine that provides protection against a broad range of influenza viruses. Therefore, highly conserved parts of the influenza A virus (IAV) were selected of which we constructed antibody and T cell inducing peptide-based vaccines. The B epitope vaccine consists of the highly conserved HA2 fusion peptide and M2e peptide coupled to a CD4 helper epitope. The T epitope vaccine comprises 25 overlapping synthetic long peptides of 26-34 amino acids, thereby avoiding restriction for a certain MHC haplotype. These peptides are derived from nucleoprotein (NP), polymerase basic protein 1 (PB1) and matrix protein 1 (M1). C57BL/6 mice, BALB/c mice, and ferrets were vaccinated with the B epitopes, 25 SLP or a combination of both. Vaccine-specific antibodies were detected in sera of mice and ferrets and vaccine-specific cellular responses were measured in mice. Following challenge, both mice and ferrets showed a reduction of virus titers in the lungs in response to vaccination. Summarizing, a peptide-based vaccine directed against conserved parts of influenza virus containing B and T cell epitopes shows promising results for further development. Such a vaccine may reduce disease burden and virus transmission during pandemic outbreaks.