Multiplex evaluation of influenza neutralizing antibodies with potential applicability to in-field serological studies

The increased number of outbreaks of H5 and H7 LPAI and HPAI viruses in poultry has major public and animal health implications. The continuous rapid evolution of these subtypes and the emergence of new variants influence the ability to undertake effective surveillance. Retroviral pseudotypes bearing influenza haemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins represent a flexible platform for sensitive, readily standardized influenza serological assays. We describe a multiplex assay for the study of neutralizing antibodies that are directed against both influenza H5 and H7 HA. This assay permits the measurement of neutralizing antibody responses against two antigenically distinct HAs in the same serum/plasma sample thus increasing the amount and quality of serological data that can be acquired from valuable sera. Sera obtained from chickens vaccinated with a monovalent H5N2 vaccine, chickens vaccinated with a bivalent H7N1/H5N9 vaccine, or turkeys naturally infected with an H7N3 virus were evaluated in this assay and the results correlated strongly with data obtained by HI assay. We show that pseudotypes are highly stable under basic cold-chain storage conditions and following multiple rounds of freeze-thaw. We propose that this robust assay may have practical utility for in-field sero-surveillance and vaccine studies in resource-limited regions worldwide

Genome sequences of four Vibrio parahaemolyticus strains isolated from the English Channel and the River Thames

This is the final version. Available from American Society for Microbiology via the DOI in this record.Data availability: Assembled and annotated genomes are publicly available within JGI IMG/M (https://img.jgi.doe.gov/) using the following taxon IDs: V. parahaemolyticus EXE V18/004 (2816332655); V. parahaemolyticus V12/024 (2816332656); V. parahaemolyticus V05/313 (2816332657) and V. parahaemolyticus V05/027 (2816332658). Read data is available on the European Nucleotide Archive under the following accession numbers: V. parahaemolyticus EXE V18/004: ERS3342146; V. parahaemolyticus V12/024: ERS3342147; V. parahaemolyticus V05/313: ERS3342148 and V. parahaemolyticus V05/027 ERS3342149.Vibrio parahaemolyticus, is the lead causative agent for seafood-borne human gastroenteritis. Whilst occurrence has traditionally been uncommon in Europe and the UK, rising sea surface temperatures have resulted in an increased prevalence. Here, we present the complete genome sequences of four novel V. parahaemolyticus strains, isolated from the UK.Natural Environment Research Council (NERC)Biotechnology and Biological Sciences Research Council (BBSRC

Bat and pig IFN-induced transmembrane protein 3 restrict cell entry by influenza virus and lyssaviruses

IFN-induced transmembrane protein 3 (IFITM3) is a restriction factor that blocks cytosolic entry of numerous viruses that utilize acidic endosomal entry pathways. In humans and mice, IFITM3 limits influenza-induced morbidity and mortality. Although many IFITM3-sensitive viruses are zoonotic, whether IFITMs function as antiviral restriction factors in mammalian species other than humans and mice is unknown. Here, IFITM3 orthologues in the microbat (Myotis myotis) and pig (Sus scrofa domesticus) were identified using rapid amplification of cDNA ends. Amino acid residues known to be important for IFITM3 function were conserved in the pig and microbat orthologues. Ectopically expressed pig and microbat IFITM3 co-localized with transferrin (early endosomes) and CD63 (late endosomes/multivesicular bodies). Pig and microbat IFITM3 restricted cell entry mediated by multiple influenza haemagglutinin subtypes and lyssavirus glycoproteins. Expression of pig or microbat IFITM3 in A549 cells reduced influenza virus yields and nucleoprotein expression. Conversely, small interfering RNA knockdown of IFITM3 in pig NPTr cells and primary microbat cells enhanced virus replication, demonstrating that these genes are functional in their species of origin at endogenous levels. In summary, we showed that IFITMs function as potent broad-spectrum antiviral effectors in two mammals – pigs and bats – identified as major reservoirs for emerging viruses

Ultra-fast intramolecular vibronic coupling revealed by RIXS and RPES maps of an aromatic adsorbate on TiO2(110)

Two-dimensional resonant inelastic x-ray scattering (RIXS) and resonant photoelectron spectroscopy (RPES) maps are presented for multilayer and monolayer coverages of an aromatic molecule (bi-isonicotinic acid) on the rutile TiO2(110) single crystal surface. The data reveals ultra-fast intramolecular vibronic coupling upon core-excitation from the N 1s orbital into the lowest unoccupied molecular orbital (LUMO) derived resonance. In the RIXS measurements this results in the splitting of the participator decay channel into a purely elastic line which disperses linearly with excitation energy, and a vibronic coupling channel at constant emission energy. In the RPES measurements the vibronic coupling results in a linear shift in binding energy of the participator channel as the excitation is tuned over the LUMO-derived resonance. Localisation of the vibrations on the molecule on the femtosecond timescale results in predominantly inelastic scattering from the core-excited state in both the physisorbed multilayer and the chemisorbed monolayer

Purification and biochemical characterization of four iron superoxide dismutases in Trypanosoma cruzi

Four superoxide dismutase (SOD) activities (SOD I, II, III, and IV) have been characterized in the epimastigote form of Trypanosoma cruzi . The total extract was subjected to two successive ammonium sulphate additions between 35 and 85%, and the resulting fraction was purified using two continuous chromatography processes (ion exchange and filtration). Enzymes were insensitive to cyanide but sensitive to hydrogen peroxide, properties characteristic of iron-containing SODs. The molecular masses of the different SODs were 20 kDa (SOD I), 60 kDa (SOD II), 50 kDa (SOD III) and 25 kDa (SOD IV), whereas the isoelectric points were 6.9, 6.8, 5.2 and 3.8, respectively. Subcellular location and digitonin experiments have shown that these SODs are mainly cytosolic, with small amounts in the low- mass organelles (SOD II and SOD I) and the mitochondrion (SOD III), where these enzymes play an important role in minimizing oxidative damage.Financial support: CGL2006-27889-E/BOS, Ministerio de Ciencia y Tecnología

Influenza hemagglutinin stem-fragment immunogen elicits broadly neutralizing antibodies and confers heterologous protection

Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. Current influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies (bnAbs), thereby limiting their efficacy. Although several bnAbs bind to the conserved stem domain of HA, focusing the immune response to this conserved stem in the presence of the immunodominant, variable head domain of HA is challenging. We report the design of a thermotolerant, disulfide-free, and trimeric HA stem-fragment immunogen which mimics the native, prefusion conformation of HA and binds conformation specific bnAbs with high affinity. The immunogen elicited bnAbs that neutralized highly divergent group 1 (H1 and H5 subtypes) and 2 (H3 subtype) influenza virus strains in vitro. Stem immunogens designed from unmatched, highly drifted influenza strains conferred robust protection against a lethal heterologous A/Puerto Rico/8/34 virus challenge in vivo. Soluble, bacterial expression of such designed immunogens allows for rapid scale-up during pandemic outbreaks

Stalking influenza by vaccination with pre-fusion headless HA mini-stem.

Inaccuracies in prediction of circulating viral strain genotypes and the possibility of novel reassortants causing a pandemic outbreak necessitate the development of an anti-influenza vaccine with increased breadth of protection and potential for rapid production and deployment. The hemagglutinin (HA) stem is a promising target for universal influenza vaccine as stem-specific antibodies have the potential to be broadly cross-reactive towards different HA subtypes. Here, we report the design of a bacterially expressed polypeptide that mimics a H5 HA stem by protein minimization to focus the antibody response towards the HA stem. The HA mini-stem folds as a trimer mimicking the HA prefusion conformation. It is resistant to thermal/chemical stress, and it binds to conformation-specific, HA stem-directed broadly neutralizing antibodies with high affinity. Mice vaccinated with the group 1 HA mini-stems are protected from morbidity and mortality against lethal challenge by both group 1 (H5 and H1) and group 2 (H3) influenza viruses, the first report of cross-group protection. Passive transfer of immune serum demonstrates the protection is mediated by stem-specific antibodies. Furthermore, antibodies indudced by these HA stems have broad HA reactivity, yet they do not have antibody-dependent enhancement activity

Infection with 2009 H1N1 influenza virus primes for immunological memory in human nose-associated lymphoid tissue, offering cross-reactive immunity to H1N1 and avian H5N1 viruses

Influenza is a highly contagious mucosal infection in the respiratory tract. 2009 pandemic H1N1 (pH1N1) virus infection resulted in substantial morbidity and mortality in humans. Little is known on whether immunological memory develops following pH1N1 infection and whether it provides protection against other virus subtypes. Enzyme-linked immunosorbent spot assay was used to analyze hemagglutinin (HA)-specific memory B cell responses after virus antigen stimulation in nasal-associated lymphoid tissues (NALT) from children and adults. Individuals with serological evidence of previous exposure to pH1N1 showed significant cross-reactive HA-specific memory B responses to pH1N1, seasonal H1N1(sH1N1) and avian H5N1(aH5N1) viruses upon pH1N1 virus stimulation. pH1N1 virus antigen elicited stronger cross-reactive memory B cell responses than sH1N1 virus. Intriguingly, aH5N1 virus also activated cross-reactive memory responses to sH1N1 and pH1N1 HAs in those who had previous pH1N1 exposure, and that correlated well with the memory response stimulated by pH1N1 virus antigen. These memory B cell responses resulted in cross-reactive neutralizing antibodies against sH1N1, 1918 H1N1 and aH5N1viruses. 2009 pH1N1 infection appeared to have primed human host with B cell memory in NALT that offers cross-protective mucosal immunity against not only H1N1 but also aH5N1 viruses. These findings may have important implications to future vaccination strategies against influenza. It will be important to induce and/or enhance such cross-protective mucosal memory B cells

Ultra-fast intramolecular vibronic coupling revealed by RIXS and RPES maps of an aromatic adsorbate on TiO2(110)

Two-dimensional resonant inelastic x-ray scattering (RIXS) and resonant photoelectron spectroscopy (RPES) maps are presented for multilayer and monolayer coverages of an aromatic molecule (bi-isonicotinic acid) on the rutile TiO2(110) single crystal surface. The data reveals ultra-fast intramolecular vibronic coupling upon core-excitation from the N 1s orbital into the lowest unoccupied molecular orbital (LUMO) derived resonance. In the RIXS measurements this results in the splitting of the participator decay channel into a purely elastic line which disperses linearly with excitation energy, and a vibronic coupling channel at constant emission energy. In the RPES measurements the vibronic coupling results in a linear shift in binding energy of the participator channel as the excitation is tuned over the LUMO-derived resonance. Localisation of the vibrations on the molecule on the femtosecond timescale results in predominantly inelastic scattering from the core-excited state in both the physisorbed multilayer and the chemisorbed monolayer