Evaluation of pre- and post-fusion Human metapneumovirus F proteins as subunit vaccine candidates in mice.

Human metapneumovirus (hMPV) is an important respiratory pathogen especially in young children and elderly subjects. Our objective was to assess the immunogenicity and protection conferred by predominant pre- and post-fusion (F) hMPV-F constructs in Balb/C mice. Immunizations without adjuvant were not immunogenic whereas alum-adjuvanted hMPV-F proteins, regardless of their conformations, generated comparable neutralizing antibody titers with undetectable pulmonary viral titers following viral challenge. In conclusion, we found no apparent advantage for mixtures of predominant pre-fusion F proteins over post-fusion conformations for hMPV vaccination in opposite to recent data obtained with the human respiratory syncytial virus.

Strain-Dependent Impact of G and SH Deletions Provide New Insights for Live-Attenuated HMPV Vaccine Development.

Human metapneumovirus (HMPV) is a major pediatric respiratory pathogen with currently no specific treatment or licensed vaccine. Different strategies to prevent this infection have been evaluated, including live-attenuated vaccines (LAV) based on SH and/or G protein deletions. This approach showed promising outcomes but has not been evaluated further using different viral strains. In that regard, we previously showed that different HMPV strains harbor distinct in vitro fusogenic and in vivo pathogenic phenotypes, possibly influencing the selection of vaccine strains. In this study, we investigated the putative contribution of the low conserved SH or G accessory proteins in such strain-dependent phenotypes and generated recombinant wild type (WT) and SH- or G-deleted viruses derived from two different patient-derived HMPV strains, A1/C-85473 and B2/CAN98-75. The ΔSH and ΔG deletions led to different strain-specific phenotypes in both LLC-MK2 cell and reconstituted human airway epithelium models. More interestingly, the ΔG-85473 and especially ΔSH-C-85473 recombinant viruses conferred significant protection against HMPV challenge and induced immunogenicity against a heterologous strain. In conclusion, our results show that the viral genetic backbone should be considered in the design of live-attenuated HMPV vaccines, and that a SH-deleted virus based on the A1/C-85473 HMPV strain could be a promising LAV candidate as it is both attenuated and protective in mice while being efficiently produced in a cell-based system.

Synergistic PA and HA mutations confer mouse adaptation of a contemporary A/H3N2 influenza virus.

The mouse is the most widely used animal model for influenza virus research. However, the susceptibility of mice to seasonal influenza virus depends on the strain of mouse and on the strain of the influenza virus. Seasonal A/H3N2 influenza viruses do not replicate well in mice and therefore they need to be adapted to this animal model. In this study, we generated a mouse-adapted A/H3N2 virus (A/Switzerland/9715293/2013 [MA-H3N2]) by serial passaging in mouse lungs that exhibited greater virulence compared to the wild-type virus (P0-H3N2). Seven mutations were found in the genome of MA-H3N2: PA(K615E), NP(G384R), NA(G320E) and HA(N122D, N144E, N246K, and A304T). Using reverse genetics, two synergistically acting genes were found as determinants of the pathogenicity in mice. First, the HA substitutions were shown to enhanced viral replication in vitro and, second, the PA-K615E substitution increased polymerase activity, although did not alter virus replication in vitro or in mice. Notably, single mutations had only limited effects on virulence in vitro. In conclusion, a co-contribution of HA and PA mutations resulted in a lethal mouse model of seasonal A/H3N2 virus. Such adapted virus is an excellent tool for evaluation of novel drugs or vaccines and for study of influenza pathogenesis.

Effect of in vitro syncytium formation on the severity of human metapneumovirus disease in a murine model.

Human metapneumovirus (HMPV) is an important cause of acute respiratory tract infections (ARTI) in children, elderly individuals and immunocompromised patients. In vitro, different HMPV strains can induce variable cytopathic effects ranging from large multinucleated syncytia to focal cell rounding. In this study, we investigated the impact of different in vitro phenotypes of two HMPV strains on viral replication and disease severity in a BALB/c mouse model. We first generated two recombinant GFP-expressing HMPV viruses: C-85473, a syncytium-inducing strain (rC-85473) belonging to the A1 subtype and CAN98-75, a focal cell rounding-inducing strain (rCAN98-75) of the B2 subtype. We subsequently exchanged the F genes of both strains to create the chimeric viruses rC-85473_F and rCAN98-75_F. We demonstrated that the F protein was the sole protein responsible for the syncytium phenotype and that viruses carrying a syncytium-inducing F protein replicated to significantly higher titers in vitro. In vivo, however, the virulence and replicative capacity of the different HMPV strains did not appear to be solely dependent on the F gene but also on the viral background, with the strains containing the C-85473 background inducing more weight loss as well as increased lung viral titers, pro-inflammatory cytokines and inflammation than strains containing the CAN98-75 background. In conclusion, the F protein is the main determinant of syncytium formation and replication kinetics in vitro, although it is not the only factor implicated in HMPV disease severity in mice.

Adjuvant effect of the human metapneumovirus (HMPV) matrix protein in HMPV subunit vaccines.

The human metapneumovirus (HMPV) fusion (F) protein is the most immunodominant protein, yet subunit vaccines containing only this protein do not confer complete protection. The HMPV matrix (M) protein induces the maturation of antigen-presenting cells in vitro. The inclusion of the M protein into an F protein subunit vaccine might therefore provide an adjuvant effect. We administered the F protein twice intramuscularly, adjuvanted with alum, the M protein or both, to BALB/c mice at 3 week intervals. Three weeks after the boost, mice were infected with HMPV and monitored for 14 days. At day 5 post-challenge, pulmonary viral titres, histopathology and cytokine levels were analysed. Mice immunized with F+alum and F+M+alum generated significantly more neutralizing antibodies than mice immunized with F only [titres of 47 ± 7 (P<0.01) and 147 ± 13 (P<0.001) versus 17 ± 2]. Unlike F only [1.6 ± 0.5 × 10(3) TCID50 (g lung)(-1)], pulmonary viral titres in mice immunized with F+M and F+M+alum were undetectable. Mice immunized with F+M presented the most important reduction in pulmonary inflammation and the lowest T-helper Th2/Th1 cytokine ratio. In conclusion, addition of the HMPV-M protein to an F protein-based vaccine modulated both humoral and cellular immune responses to subsequent infection, thereby increasing the protection conferred by the vaccine.

Randomized controlled ferret study to assess the direct impact of 2008-09 trivalent inactivated influenza vaccine on A(H1N1)pdm09 disease risk.

During spring-summer 2009, several observational studies from Canada showed increased risk of medically-attended, laboratory-confirmed A(H1N1)pdm09 illness among prior recipients of 2008-09 trivalent inactivated influenza vaccine (TIV). Explanatory hypotheses included direct and indirect vaccine effects. In a randomized placebo-controlled ferret study, we tested whether prior receipt of 2008-09 TIV may have directly influenced A(H1N1)pdm09 illness. Thirty-two ferrets (16/group) received 0.5 mL intra-muscular injections of the Canadian-manufactured, commercially-available, non-adjuvanted, split 2008-09 Fluviral or PBS placebo on days 0 and 28. On day 49 all animals were challenged (Ch0) with A(H1N1)pdm09. Four ferrets per group were randomly selected for sacrifice at day 5 post-challenge (Ch+5) and the rest followed until Ch+14. Sera were tested for antibody to vaccine antigens and A(H1N1)pdm09 by hemagglutination inhibition (HI), microneutralization (MN), nucleoprotein-based ELISA and HA1-based microarray assays. Clinical characteristics and nasal virus titers were recorded pre-challenge then post-challenge until sacrifice when lung virus titers, cytokines and inflammatory scores were determined. Baseline characteristics were similar between the two groups of influenza-naïve animals. Antibody rise to vaccine antigens was evident by ELISA and HA1-based microarray but not by HI or MN assays; virus challenge raised antibody to A(H1N1)pdm09 by all assays in both groups. Beginning at Ch+2, vaccinated animals experienced greater loss of appetite and weight than placebo animals, reaching the greatest between-group difference in weight loss relative to baseline at Ch+5 (7.4% vs. 5.2%; p = 0.01). At Ch+5 vaccinated animals had higher lung virus titers (log-mean 4.96 vs. 4.23pfu/mL, respectively; p = 0.01), lung inflammatory scores (5.8 vs. 2.1, respectively; p = 0.051) and cytokine levels (p>0.05). At Ch+14, both groups had recovered. Findings in influenza-naïve, systematically-infected ferrets may not replicate the human experience. While they cannot be considered conclusive to explain human observations, these ferret findings are consistent with direct, adverse effect of prior 2008-09 TIV receipt on A(H1N1)pdm09 illness. As such, they warrant further in-depth investigation and search for possible mechanistic explanations.

Modulation of protease activated receptor 1 influences human metapneumovirus disease severity in a mouse model.

Human metapneumovirus (hMPV) infection causes acute respiratory tract infections (RTI) which can result in hospitalization of both children and adults. To date, no antiviral or vaccine is available for this common viral infection. Immunomodulators could represent an interesting strategy for the treatment of severe viral infection. Recently, the role of protease-activated receptors (PAR) in inflammation, coagulation and infection processes has been of growing interest. Herein, the effects of a PAR1 agonist and a PAR1 antagonist on hMPV infection were investigated in BALB/c mice. Intranasal administration of the PAR1 agonist resulted in increased weight loss and mortality of infected mice. Conversely, the PAR1 antagonist was beneficial to hMPV infection by decreasing weight loss and clinical signs and by significantly reducing pulmonary inflammation, pro-inflammatory cytokine levels (including IL-6, KC and MCP-1) and recruitment of immune cells to the lungs. In addition, a significant reduction in pulmonary viral titers was also observed in the lungs of PAR1 antagonist-treated mice. Despite no apparent direct effect on virus replication during in vitro experiments, an important role for PAR1 in the regulation of furin expression in the lungs was shown for the first time. Further experiments indicated that the hMPV fusion protein can be cleaved by furin thus suggesting that PAR1 could have an effect on viral infectivity in addition to its immunomodulatory properties. Thus, inhibition of PAR1 by selected antagonists could represent an interesting strategy for decreasing the severity of paramyxovirus infections.

Autofluorescence as a viability marker for detection of bacterial spores.

Recent biological terrorism events have indicated that bacterial spores such as Bacillus anthracis are real threat agents. Real time detection of biological agents is possible with the use of an ultraviolet Fluorescent Aerodynamic Particle Sizer (FLAPS) that measures particles' intrinsic fluorescence. It is important to know whether intrinsic fluorescence could be used to estimate agents' viability. Two categories of Bacillus spore populations can be differentiated by the intensity of intrinsic fluorescence emitted by ultraviolet (UV) stimulation : autofluorescent and non-autofluorescent. This study was performed to determine whether intensity of autofluorescence correlates with spore viability. Spores were analyzed using flow cytometer (equipped with a cell sorter) to mimic optical properties of FLAPS. Autofluorescent and non-autofluorescent spores were sorted according to the intensity of autofluorescence emitted following UV stimulation. Culturability, membrane integrity, membrane potential and dipicolinic acid (DPA) content were assessed. Autofluorescent spores were 1.7 times more culturable than the corresponding non-autofluorescent population. Moreover, a small proportion of autofluorescent spores exhibited extracellular membrane damages. Autofluorescent spores also showed higher membrane potential activity and contained higher levels of DPA. In conclusion, this study documents that the overall viability potential of bacterial spores can be assessed by UV flow cytometry used in the FLAPS technology.

Expression of FcgammaRIII (CD16) on human peripheral blood eosinophils increases in allergic conditions.

BACKGROUND: Blood eosinophils have mRNA for FcgammaRIIIB (CD16) but no or minimal spontaneous CD16 expression. Because IFN-gamma and chemotactic factors induce eosinophil CD16 expression in vitro, we postulated that blood eosinophils could express CD16. OBJECTIVE: Blood of nonallergic controls and subjects with allergic rhinitis, allergic and nonallergic asthma, or hypereosinophilia of various etiologies were analyzed for leukocyte CD16 surface expression. METHODS: CD16(+) eosinophils were identified on the basis of physico-optic characteristics, major basic protein, CD49b expression, and sorting by flow cytometry and microscope examination. RESULTS: Subjects with allergic rhinitis and subjects with asthma had higher median percentages of CD16(+) eosinophils (8.1% [1% to 48.6%] and 7.3% [1.4% to 31.1%], respectively) than nonallergic controls and nonallergic asthmatics (3% [0% to 11%] and 4.6% [2.9% to 5.1%], respectively). In subjects with hypereosinophilia, CD16(+) eosinophils were increased only in a case of drug allergy. When subjects with mild allergic asthma were challenged with a relevant aeroallergen, blood CD16(+) eosinophils further increased during or after the late-phase response (6 to 48 hours after challenge; mean +/- SEM, 9.4% +/- 2.5% to 20.0% +/- 3.0%). CD16(+) eosinophils expressed more IL-5 receptor but less CD11b and IL-12p35 than did CD16(-) eosinophils. CONCLUSION: Upregulation of blood CD16(+) eosinophils in allergic conditions and its association with a modified phenotype suggest that CD16 receptor could play a role in eosinophil activation in allergy.