Highly pathogenic avian influenza is an on-going problem in poultry and a potential human pandemic threat. Pandemics occur suddenly and vaccine production must be fast and effective to be of value in controlling the spread of the virus. In this study we evaluated the potential of a recombinant protein from the extracellular domain of an H5 hemagglutinin protein produced in a yeast expression system to act as an effective vaccine. Protein production was efficient, with up to 200 mg purified from 1 L of culture medium. We showed that the deletion of the multibasic cleavage site from the protein improves oligomerization and, consequentially, its immunogenicity. We also showed that immunization with this deleted protein protected chickens from challenge with a highly pathogenic avian influenza H5N1 virus. Our results suggest that this recombinant protein produced in yeast may be an effective vaccine against H5N1 virus in poultry.
Highly pathogenic avian influenza viruses cause severe disease and huge economic losses in domestic poultry and might pose a serious threat to people because of the high mortality rates in case of an accidental transmission to humans. The main goal of this work was to evaluate the immune responses and hemagglutination inhibition potential elicited by a combined DNA/recombinant protein prime/boost vaccination compared to DNA/DNA and protein/protein regimens in chickens. A plasmid encoding hemagglutinin (HA) from the A/swan/Poland/305-135V08/2006 (H5N1) virus, or the recombinant HA protein produced in Pichia pastoris system, both induced H5 HA-specific humoral immune responses in chickens. In two independent experiments, anti-HA antibodies were detected in sera collected two weeks after the first dose and the response was enhanced by the second dose of a vaccine, regardless of the type of subunit vaccine (DNA or recombinant protein) administered. The serum collected from chickens two weeks after the second dose was characterized by three types of assays: indirect ELISA, hemagglutination inhibition (HI) and a diagnostic test based on H5 antibody competition. Although the indirect ELISA failed to detect superiority of any of the three vaccine regimens, the other two tests clearly indicated that priming of chickens with the DNA vaccine significantly enhanced the protective potential of the recombinant protein vaccine produced in P. pastoris.
Hemagglutinin glycoprotein (HA) is a principle influenza vaccine antigen. Recombinant HA-based vaccines become a potential alternative for traditional approach. Complexity and variation of HA N-glycosylation are considered as the important factors for the vaccine design. The number and location of glycan moieties in the HA molecule are also crucial. Therefore, we decided to study the effect of N-glycosylation pattern on the H5 antigen structure and its ability to induce immunological response. We also decided to change neither the number nor the position of the HA glycosylation sites but only the glycan length. Two variants of the H5 antigen with high mannose glycosylation (H5hm) and with low-mannose glycosylation (H5Man5) were prepared utilizing different Pichia strains. Our structural studies demonstrated that only the highly glycosylated H5 antigen formed high molecular weight oligomers similar to viral particles. Further, the H5hm was much more immunogenic for mice than H5Man5. In summary, our results suggest that high mannose glycosylation of vaccine antigen is superior to the low glycosylation pattern. Our findings have strong implications for the recombinant HA-based influenza vaccine design.
Negocjacje stanowią ważny aspekt życia społecznego oraz gospodarczego. Wprawnym negocjatorom możemy zawdzięczać koniec wojny albo jej zapobiegnięcie, a także wiele innych korzystnych rozwiązań różnego rodzaju konfliktów. Wraz z początkiem pandemii niemożność spotkania się partnerów negocjacyjnych na żywo, twarzą w twarz, skłoniło przedsiębiorstwa, ale też inne podmioty, do sięgnięcia po aplikacje umożliwiające prowadzenie negocjacji w formule zdalnej, czyli na odległość. Celem artykułu jest zidentyfikowanie kluczowych czynników sukcesu negocjacji zdalnych, by wskazać dobre praktyki w tym zakresie, a także zidentyfikować te obszary, którymi różnią się negocjacje stacjonarne od zdalnych. Przeprowadzone badanie miało charakter jakościowy i polegało na badaniu ankietowym próby celowej. Wybrano do niej osoby, dla których negocjacje stanowią istotny element życia zawodowego. Główny wniosek płynący z badań skłania do stwierdzenia, że czynniki sukcesu negocjacji zdalnych są zasadniczo takie same, jak stacjonarnych, przy czym większej staranności należy dołożyć w kwestii technologii – zarówno własnych umiejętności w zakresie obsługi sprzętu, jak też zapewnienia odpowiedniej jakości infrastruktury.
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