Bacterially expressed full length Hemagglutinin of Avian Influenza Virus H5N1 forms oligomers and exhibits hemagglutination.

Avian influenza poses a significant global health threat, with the potential for widespread pandemics and devastating consequences. Hemagglutinin (HA), a critical surface glycoprotein of influenza viruses, plays a pivotal role in viral entry and serves as a primary target for subunit vaccine development. In this study, we successfully cloned, expressed, and purified hemagglutinin from the circulating strain of H5N1 influenza virus using a robust molecular biology approach. The cloning process involved insertion of the synthetic HA gene into the pET21b vector, confirmed through double digestion and sequencing. SDS-PAGE analysis confirmed the presence of the expected 60 kDa protein band post-induction. Following expression, the protein was subjected to purification via Ni-NTA affinity chromatography, yielding pure protein fractions. Native PAGE analysis confirmed the protein's oligomeric forms, essential for optimal antigenicity. Western blot analysis further validated protein identity using anti-His and anti-HA antibodies. MALDI-TOF analysis confirmed the protein's sequence integrity, while hemagglutination assay demonstrated its biological activity in binding to N-acetyl neuraminic acid. These findings underscore the potential of recombinant hemagglutinin as a valuable antigen for diagnosis and biochemical assays as well as for vaccine development against avian influenza. In conclusion, this study represents a critical guide for bacterial production of H5N1 HA, which can be a cost-effective and simpler strategy compared to mammalian protein expression. Further research into optimizing vaccine candidates and production methods will be essential in combating the ongoing threat of avian influenza pandemics.

Lead Optimization of Dehydroemetine for Repositioned Use in Malaria.

Drug repositioning offers an effective alternative to de novo drug design to tackle the urgent need for novel antimalarial treatments. The antiamoebic compound emetine dihydrochloride has been identified as a potent in vitro inhibitor of the multidrug-resistant strain K1 of Plasmodium falciparum (50% inhibitory concentration [IC50], 47 nM ± 2.1 nM [mean ± standard deviation]). Dehydroemetine, a synthetic analogue of emetine dihydrochloride, has been reported to have less-cardiotoxic effects than emetine. The structures of two diastereomers of dehydroemetine were modeled on the published emetine binding site on the cryo-electron microscopy (cryo-EM) structure with PDB code 3J7A (P. falciparum 80S ribosome in complex with emetine), and it was found that (-)-R,S-dehydroemetine mimicked the bound pose of emetine more closely than did (-)-S,S-dehydroisoemetine. (-)-R,S-dehydroemetine (IC50 71.03 ± 6.1 nM) was also found to be highly potent against the multidrug-resistant K1 strain of P. falciparum compared with (-)-S,S-dehydroisoemetine (IC50, 2.07 ± 0.26 µM), which loses its potency due to the change of configuration at C-1'. In addition to its effect on the asexual erythrocytic stages of P. falciparum, the compound exhibited gametocidal properties with no cross-resistance against any of the multidrug-resistant strains tested. Drug interaction studies showed (-)-R,S-dehydroemetine to have synergistic antimalarial activity with atovaquone and proguanil. Emetine dihydrochloride and (-)-R,S-dehydroemetine failed to show any inhibition of the hERG potassium channel and displayed activity affecting the mitochondrial membrane potential, indicating a possible multimodal mechanism of action.