Although effective antigenic epitopes for HPIV vaccine remain elusive (Henrickson, 2003), a monoclonal antibody targeting the stalk region of HPIV2-HN has been shown to have a profound inhibitory activity against viral infection (Yuasa et al., 1995). model. Our results provide the first evidence that intranasal co-administration of OML-encapsulated HN with the poly(I:C) adjuvant augments the viral-specific immunity against HPIV3. test: ** 0.01, * 0.05) We next measured the levels of serum IgG and nasal wash IgA in each individual mouse by ELISA and immunoblotting (Figures ?Figures3A3ACC). Mice immunized with OML-HN (1 g) plus Poly(I:C) exhibited prominent induction of HN-specific IgG (Figures ?Figures3A3A,?,CC). HN-specific IgA in nasal wash fluid was most prominently induced in mice with OML-HN (1 g) plus Inogatran Poly(I:C) compared to other groups (Physique ?Figure3B3B). Interestingly, the induction of the HN-specific IgA was higher in mice that were immunized with the lower amount of antigens, OML-HN (0.1 g) plus Poly(I:C) (Figure ?Physique3B3B). Open in a separate window Physique 3 Measurement of HPIV3-HN-specific serum IgG and nasal wash IgA. (A) The levels of HN-specific IgG in the serum of each immunized mouse on day 21 after the third immunization were determined by ELISA. (B) The levels of HN-specific IgA in nasal wash fluid on day 28 after the third immunization were determined by ELISA. Each bar represents the mean SE (test: * 0.05). (C) Immunoblot analysis of recombinant HN proteins in mouse sera. Recombinant GST or GST-HN proteins were subjected to SDS-PAGE followed by the immunoblotting with the indicated serum. EPITOPE MAPPING OF INDUCED ANTIBODIES We next determined the region of HN that was recognized by the HN-specific serum IgG produced by the mice that were immunized with OML-HN (1 g) plus Poly(I:C). Three domain name mutants of HPIV3-HN, the N-terminal region (1-190), the middle region (168-408) and C-terminal region (400-572) were synthesized using the wheat germ cell-free system (Figure ?Physique4A4A left) and protein production was confirmed by SDS-PAGE (Physique ?Figure4A4A right). Based on ELISA analysis, all of the serum samples contained HN-specific antibodies that had high reactivity to the N-terminal region (Figure ?Physique4B4B). Open in a separate window Physique 4 Anti-infectious activity of mouse serum. (A,B) Epitope mapping of antibodies induced in the immunized mouse. We selected arbitrary three representative sera from mice immunized with OML-HN (1 g) plus Poly(I:C) that exhibited the IL1R highest HN-specific IgG induction (#1C#3). The full-length and three deletion mutants of GST-HN were produced using the wheat germ cell-free system. These purified proteins were separated by SDS-PAGE and visualized using CBB staining (A). Using the recombinant HN proteins, the target region of the three sera (#1C#3) was analyzed by ELISA (B). (C) Schematic representation of the experimental procedure of infection-inhibitory assay (left panel). Immunized mouse sera (#1C#3) were tested for this assay. An OML-empty-treated mouse serum was used as a control. The anti-infection ability was measured using quantitative real-time PCR for HPIV3-HN mRNA. Each bar represents the mean SE of two impartial experiments as normalized by control serum (test: * 0.05). EFFECT OF OML-HN VACCINE ON HPIV3 Contamination and baculovirus systems, the wheat Inogatran germ cell-free system is beneficial for the rapid and efficient preparation of high-quality proteins (Endo and Sawasaki, 2006). Moreover, this cell-free system is suitable for the generation of toxic viral proteins for immunization and beneficial for the purification of naturally folded proteins, as well Inogatran as scalability. This system, however, may not be cost-effective Inogatran for preparing large amounts of viral antigens for vaccine development. Therefore, efforts were made to reduce the amount of antigen needed vaccination. Herein, we utilized a OML and Poly(I:C) vaccination strategy in an attempt to reduce the amount of antigen required. OML is usually a lipid vesicle that has mannose on its surface, which aids in efficient targeting to APCs (Shimizu et al., 2007; Nishimura et al., 2013). In a previous report, antigenic.