BackgroundThe highly pathogenic avian influenza (HPAI) H5N1 virus continues to cause disease in poultry and humans. The hemagglutinin (HA) envelope protein is the primary target for subunit vaccine development.Methodology/Principal FindingsWe used baculovirus-insect cell expression to obtain trimeric recombinant HA (rHA) proteins from two HPAI H5N1 viruses. We investigated trimeric rHA protein immunogenicity in mice via immunizations, and found that the highest levels of neutralizing antibodies resulted from coupling with a PELC/CpG adjuvant. We also found that the combined use of trimeric rHA proteins with (a) an inactivated H5N1 vaccine virus, or (b) a recombinant adenovirus encoding full-length HA sequences for prime-boost immunization, further improved antibody responses against homologous and heterologous H5N1 virus strains. Data from cross-clade prime-boost immunization regimens indicate that sequential immunization with different clade HA antigens increased antibody responses in terms of total IgG level and neutralizing antibody titers.Conclusion/SignificanceOur findings suggest that the use of trimeric rHA in prime-boost vaccine regimens represents an alternative strategy for recombinant H5N1 vaccine development.

Current egg-based influenza vaccine production technology can't promptly meet the global demand during an influenza pandemic as shown in the 2009 H1N1 pandemic. Moreover, its manufacturing capacity would be vulnerable during pandemics caused by highly pathogenic avian influenza viruses. Therefore, vaccine production using mammalian cell technology is becoming attractive. Current influenza H5N1 vaccine strain (NIBRG-14), a reassortant virus between A/Vietnam/1194/2004 (H5N1) virus and egg-adapted high-growth A/PR/8/1934 virus, could grow efficiently in eggs and MDCK cells but not Vero cells which is the most popular cell line for manufacturing human vaccines. After serial passages and plaque purifications of the NIBRG-14 vaccine virus in Vero cells, one high-growth virus strain (Vero-15) was generated and can grow over 108 TCID50/ml. In conclusion, one high-growth H5N1 vaccine virus was generated in Vero cells, which can be used to manufacture influenza H5N1 vaccines and prepare reassortant vaccine viruses for other influenza A subtypes.

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Cross-presentation by DCs is the major mechanism by which exogenous antigens activate CTLs. However, the mechanisms of entry and presentation of vaccine peptides by DCs remain unclear. In this study, we determined that the mechanisms of antigen presentation differed between nonlipidated and monopalmitoylated peptide antigens. We found that a nonlipidated long peptide could be taken up by DCs and that the peptide could be colocalized with early endosomes. The uptake of nonlipidated peptides by DCs was inhibited at low temperatures or by the depolymerization of actin filaments or microtubules. In contrast, lipidated peptides were internalized by DCs at low temperatures, and internalization was not inhibited when actin filaments or microtubules were depolymerized. Moreover, lipidated peptide, but not nonlipidated peptide, was internalized by nonphagocytic Jurkat cells. The endosomal/lysosomal and proteasomal degradation pathways were necessary for nonlipidated presentation leading to the activation of CD8(+) T cells, but the proteasomal degradation pathway alone was sufficient to process lipidated peptides for MHC class I presentation. We further found that lipidated peptides could enhance peptide-specific T cell responses in vitro and in vivo and induced stronger antitumor responses than nonlipidated peptides. Taken together, our results demonstrate that DCs present lipidated peptides through an endocytosis-independent pathway to promote strong anti-tumor effects in vivo.

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