Environmental Stability of Swine and Human Pandemic Influenza Viruses in Water under Variable Conditions of Temperature, Salinity, and pH.

UNLABELLED: The movement of influenza A viruses (IAVs) from wild bird reservoirs to domestic animals and humans is well established, but the transmission mechanisms that facilitate efficient movement across and within these host populations are not fully defined. Although predominant routes of transmission vary between host populations, the extent of environmental stability needed for efficient IAV transmission also may vary. Because of this, we hypothesized that virus stability would differ in response to varied host-related transmission mechanisms; if correct, such phenotypic variation might represent a potential marker for the emergence of novel animal or human influenza viruses. Here, the objective was to evaluate the ability of eight swine and six human IAV isolates to remain infective under various pH, temperature, and salinity conditions using a preestablished distilled water system. Swine and human viruses persisted longest at near-neutral pH, at cold temperatures, or under "freshwater" conditions. Additionally, no significant differences in persistence were observed between pandemic and nonpandemic IAVs. Our results indicate that there have been no apparent changes in the environmental stability of the viruses related to host adaptation. IMPORTANCE: This study assessed the environmental stability of eight swine and six human influenza A viruses (IAVs), including viruses associated with the 2009 H1N1 pandemic, in a distilled water system. The important findings of this work are that IAV persistence can be affected by environmental variables and that no marked changes were noted between human and swine IAVs or between pandemic and nonpandemic IAVs.

Influenza Research & Development - GTCBio's Third Annual Conference (July 9-11 - Boston, Massachusetts, USA, 2014).

The Third Influenza Research and Development Conference organized by GTCBio was convened as part of their Infectious Disease World Summit 2014, which also included the 12th Vaccines Research & Development: All Things Considered and 11th Anti-infectives Partnering and Deal-Making Conferences. The Influenza Research and Development Conference consisted of eight sessions: 'Development of Influenza Vaccines' (held jointly with Vaccines Research & Development), 'Antivirals and Therapeutics', 'Role of T-Cells', 'Regulatory Guidance - Clinical Trial Design & Efficacy Trials', 'Role of Antibodies' and 'Innate Immunity and Infection'. The conference also convened a panel at the end of the second day to discuss 'Balancing Science & the First Amendment', as well as a session at the end of day 3 for oral presentations selected from exemplary submitted abstracts. The overall scope of the conference addressed the need for improved influenza vaccines, approaches for assessing vaccine efficacy, novel vaccination strategies and novel antiviral drugs. Presentations by academics and industry provided a good balance of cutting-edge research with preclinical, clinical and post-licensure studies. Interventions for human seasonal, zoonotic and pandemic influenza viruses were also discussed.

Domestic cats are susceptible to infection with low pathogenic avian influenza viruses from shorebirds.

Domestic cats are susceptible to infection with highly pathogenic avian influenza virus H5N1, resulting in pneumonia and in some cases, systemic spread with lesions in multiple organ systems. Recent transmission of the 2009 pandemic H1N1 influenza virus from humans to cats also resulted in severe pneumonia in cats. Data regarding the susceptibility of cats to other influenza viruses is minimal, especially regarding susceptibility to low pathogenic avian influenza viruses from wild birds, the reservoir host. In this study, the authors infected 5-month-old cats using 2 different North American shorebird avian influenza viruses (H1N9 and H6N4 subtypes), 3 cats per virus, with the goal of expanding the understanding of avian influenza virus infections in this species. These viruses replicated in inoculated cats based on virus isolation from the pharynx in 2 cats, virus isolation from the lung of 1 cat, and antigen presence in the lung via immunohistochemistry in 2 cats. There was also seroconversion and lesions of patchy bronchointerstitial pneumonia in all of the cats. Infection in the cats did not result in clinical disease and led to variable pharyngeal viral shedding with only 1 of the viruses; virus was localized in the alveolar epithelium via immunohistochemistry. These findings demonstrate the capacity of wild bird influenza viruses to infect cats, and further investigation is warranted into the pathogenesis of these viruses in cats from both a veterinary medical and public health perspective.

A humanized anti-M2 scFv shows protective in vitro activity against influenza.

M2 is one of the most conserved influenza proteins, and has been widely prospected as a potential universal vaccine target, with protection predominantly mediated by antibodies. In this paper we describe the creation of a humanized single chain Fv from 14C2, a potent monoclonal antibody against M2. We show that the humanized scFv demonstrates similar activity to the parental mAb: it is able to recognize M2 in its native context on cell surfaces and is able to show protective in vitro activity against influenza, and so represents a potential lead antibody candidate for universal prophylactic or therapeutic intervention in influenza.

Assessment of the role of determinant selection in genetic control of the immune response to insulin in H-2b mice.

The immune response to insulin is regulated by MHC class II genes. Immune response (Ir) gene-linked low responsiveness to protein Ags can be mediated by the low affinity of potential antigenic determinants for MHC molecules (determinant selection) or by the influence of MHC on the functional T cell repertoire. Strong evidence exists that determinant selection plays a key role in epitope immunodominance and Ir gene-linked unresponsiveness. However, the actual measurement of relative MHC-binding affinities of all potential peptides derived from well-characterized model Ags under Ir gene regulation has been very limited. We chose to take advantage of the simplicity of the structure of insulin to study the mechanism of Ir gene control in H-2b mice, which respond to beef insulin (BINS) but not pork insulin (PINS). Peptides from these proteins, including the immunodominant A(1-14) determinant, were observed to have similar affinities for purified IAb in binding experiments. Functional and biochemical experiments suggested that PINS and BINS are processed with similar efficiency. The T cell response to synthetic pork A(1-14) was considerably weaker than the response to the BINS peptide. We conclude that the poor immunogenicity of PINS in H-2b mice is a consequence of the T cell repertoire rather than differences in processing and presentation.

Transporters associated with antigen processing (TAP)-independent presentation of soluble insulin to alpha/beta T cells by the class Ib gene product, Qa-1(b).

T cell hybridomas isolated from nonresponder H-2(b) mice immunized with pork insulin were stimulated by insulin in the presence of major histocompatibility complex (MHC)-unmatched antigen presenting cells. The restriction element used by these CD4(-) T cells was mapped to an oligomorphic MHC class Ib protein encoded in the T region and identified as Qa-1(b) using transfectants. The antigenic determinant was localized to the insulin B chain, and experiments with truncated peptides suggested that it is unexpectedly long, comprising most or all of the 30 amino acid B chain. The antigen processing pathway used to present insulin to the Qa-1(b)- restricted T cells does not require transporters associated with antigen processing (TAP), and it is inhibited by chloroquine. A wide variety of cell lines from different tissues efficiently present soluble insulin to Qa-1(b)-restricted T cells, and insulin presentation is not enhanced by phagocytic stimuli. Our results demonstrate that Qa-1(b) can function to present exogenous protein to T cells in a manner similar to MHC class II molecules. Therefore, this class Ib protein may have access to a novel antigen processing pathway that is not available to class Ia molecules.

An insulin peptide that binds an alternative site in class II major histocompatibility complex.

We report that a peptide from the B chain of insulin, B(10-30), binds with high affinity to multiple class II proteins, including IAb,d,k, IEd,k, and DR1. The ability of B(10-30) to inhibit the binding of other peptide antigens to class II does not correlate with its affinity for class II. B(10-30) only weakly inhibits the binding of antigenic peptides. Conversely, peptides with high affinity for the peptide-binding groove of various class II proteins do not inhibit B(10-30) binding. The rate of association of B(10-30) with class II is unusually rapid, approaching saturation in 1-2 h compared with 1-2 d for classical peptide antigens in the same conditions. The dissociation rate is also relatively rapid. The B(10-30) peptide inhibits the binding of the super-antigen staphylococcal enterotoxin B (SEB) to IAk. It also inhibits SEB-mediated T cell activation. These observations support the conclusion that B(10-30) binds to a site outside the peptide-binding groove. Our findings indicate that short-lived peptide-class II complexes can be formed through interactions involving the SEB-binding site and raise the possibility that alternative complexes may serve as T cell receptor ligands.

A europium fluoroimmunoassay for measuring binding of antigen to class II MHC glycoproteins.

A dissociation-enhanced lanthanide fluoroimmunoassay employing europium-streptavidin and time-resolved fluorimetry was developed to measure binding of biotin-labeled peptides to class II MHC proteins. Binding of biotin-peptides as measured by this assay was saturable and inhibited in the presence of unlabeled peptide. Background fluorescence was minimal and there was a direct relationship between signal and biotin-peptide/class II complex concentration from 1.3 pmol to less than 1 fmol total class II. The sensitivity of the assay and the ability to selectively capture specific class II proteins from detergent lysates of cells with solid phase mAb made it possible to measure formation peptide/class II complexes in live APC cultured with biotin-labeled insulin. This assay is expected to be useful for routine measurement of peptide/class II binding and biochemical analysis of Ag processing events.

Characterization of an immediate-early gene induced in adherent monocytes that encodes I kappa B-like activity.

We have cloned a group of cDNAs representing mRNAs that are rapidly induced following adherence of human monocytes. One of the induced transcripts (MAD-3) encodes a protein of 317 amino acids with one domain containing five tandem repeats of the cdc10/ankyrin motif, which is 60% similar (46% identical) to the ankyrin repeat region of the precursor of NF-kappa B/KBF1 p50. The C-terminus has a putative protein kinase C phosphorylation site. In vitro translated MAD-3 protein was found to specifically inhibit the DNA-binding activity of the p50/p65 NF-kappa B complex but not that of the p50/p50 KBF1 factor or of other DNA-binding proteins. The MAD-3 cDNA encodes an I kappa B-like protein that is likely to be involved in regulation of transcriptional responses to NF-kappa B, including adhesion-dependent pathways of monocyte activation.