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The severe acute respiratory syndrome (SARS) is a contagious disease that killed hundreds and sickened thousands of people worldwide between November 2002 and July 2003. The nucleocapsid (N) protein of the coronavirus responsible for this disease plays a critical role in viral assembly and maturation and is of particular interest because of its potential as an antiviral target or vaccine candidate. Refolding of SARS N-protein during production and purification showed the presence of two additional protein bands by SDS-PAGE. Mass spectroscopy (MALDI, SELDI, and LC/MS) confirmed that the bands are proteolytic products of N-protein and the cleavage sites are four SR motifs in the serine-arginine-rich region-sites not suggestive of any known protease. Furthermore, results of subsequent testing for contaminating protease(s) were negative: cleavage appears to be due to inherent instability and/or autolysis. The importance of N-protein proteolysis to viral life cycle and thus to possible treatment directions are discussed.

CD40L, a key regulator of the immune system, was studied as both a targeting ligand and a molecular adjuvant in nucleoprotein (NP)-based host defense against influenza in mouse models with different genetic backgrounds. Adenoviral vectors secreting NP-CD40L fusion protein (denoted as rAd-SNP40L) afforded full protection of immunocompetent and immunocompromised mice (CD40L−/− and CD4−/−) against lethal influenza infection. Mechanistically, rAd-SNP40L preferentially induced early and persistent B cell germinal center formation, and accelerated Ig isotype-switching and Th1-skewed, NP-specific Ab response. Moreover, it drastically augmented primary and memory NP-specific CTL activity and polyfunctional CD8+ T cells. The markedly enhanced nonneutralizing Abs and CTLs significantly reduced viral burdens in the lungs of mice upon lethal virus challenge. Data generated from CD40L−/− and CD4−/− mice revealed that the protection was indeed CD40L mediated but CD4+ T cell independent, demonstrating the viability of the fusion Ags in protecting immunodeficient hosts. Notably, a single dose of rAd-SNP40L completely protected mice from lethal viral challenge 4 mo after immunization, representing the first report, to our knowledge, on NP in conjunction with a molecular adjuvant inducing a robust and long-lasting memory immune response against influenza. This platform is characterized by an increased in vivo load of CD40-targeted Ag upon the secretion of the fusion protein from adenovirus-infected cells and may represent a promising strategy to enhance the breadth, durability, and potency of Ag-specific immune responses.

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The fusion peptide of influenza virus hemagglutinin (HA) has a critical role in mediating the entry of the virus into the cells and is also the only universally conserved sequence in the HAs of all strains of influenza A and influenza B viruses. Therefore, it could be an attractive target for new vaccine development and a potency marker for existing influenza vaccines. The fusion peptide epitope is hidden inside the HA proteins, making it inaccessible for quantitative antibody binding. Our simple slot blot protocol highlights pre-treatment of HA samples with moderate concentrations of denaturant to maximally expose the fusion peptide on the protein surface, followed by detection using universal antibodies targeting the fusion peptide. The method is highly reliable, inexpensive and easy to follow. The entire procedure takes only 5 h and can be applied to the quantitative determination of virtually all influenza viral HAs using a single antibody targeting the fusion peptide.