For passive transfer experiments, mice were bled 2 wk after the last immunization with KLH or LAH-KLH or 3 wk after infection with PR8 computer virus or A/Hong Kong/1/1968 computer virus. induction of broadly active anti-hemagglutinin antibodies. These results provide proof of concept for an HA2-centered influenza vaccine that could diminish the threat of pandemic influenza disease and generally reduce the significance of influenza viruses as human being pathogens. and and and and and and = five BALB/c mice per group. Because of variations in pathogenicity, survival was defined as 20% excess weight loss for X31 (H3) and PR8 (H1) viruses and 30% excess weight loss for VN/2004 (H5) computer virus. Next, immunized mice were challenged with additional computer virus subtypes that cause human being influenza disease but that belong to a phylogenetic class unique from H3 subtype viruses (6). Mice were infected with 500 pfu [10C15 occasions the dose lethal to 50% of mice (MLD50)] of PR8 (a mouse-adapted H1 computer virus) or with 500 pfu of an H5 highly pathogenic avian influenza computer virus modified to remove the polybasic cleavage site in the viral hemagglutinin (12). Vaccination with the LAH-KLH conjugate was protecting against excess weight loss caused by H1 and H5 influenza disease to a highly significant degree on virtually every day during illness. Vaccinated mice infected with PR8 showed a significant delay in kinetics of excess weight loss, and 60% of vaccinated mice infected with the H5 avian computer virus survived lethal challenge to 10 d postinfection (Fig. 3 and 0.001). (and = 0.0009) or the H1 virus (= 0.0008) (Fig. 4 and and and Table 1). Because the LAH structure is present in the HA2 protein, the broad reactivity seen in the LAH-KLH antiserum must be a consequence of the manner in which the LAH is definitely offered as an antigen within the conjugate complex. Removal of immunodominant regions of the HA2 protein may cause the LAH-KLH vaccine to induce a more focused anti-LAH immune response that mediates broad reactivity Menaquinone-7 between hemagglutinin subtypes. Alternately, the induction of broadly reactive antibody may be a consequence of anchoring the LAH in the C terminus to a carrier protein, thus rendering immunogenic regions of the LAH that normally are antigenically silent in the context of the intact HA2 protein. Table 1. Summary of ELISA data in Fig. 5 and em C /em ), or influenza computer virus vaccine [FLUVIRON (R), from BEI Resources] purified surface antigen (Novartis Vaccines) in PBS over night at 4 C. Plates were clogged for 30 min at space heat with 1% BSA/PBS Menaquinone-7 and were washed twice with PBS/0.025% Tween. Antibodies, antiserum, or serum from individuals vaccinated with the 2008C2009 TIV were serially diluted in 1% BSA/PBS and added to the Menaquinone-7 plate followed by 3-h incubation at 37 C. An anti-Flag antibody (Sigma) was used like a positive control in wells coated with the LAH-KLH conjugate. Plates were washed three times, and anti-mouse alkaline phosphatase (AP) (Southern Biotech) diluted Menaquinone-7 1:2,000 was added to wells followed by 3-h incubation at 37 C. For human being sera, anti-human IgG (Fc spscific)-AP (Sigma) antibody was used at 1:500 dilution. Anti-rabbit Ig-AP (Southern Biotech) at 1:500 dilution was used as the secondary for the anti-Flag antibody. P-nitrophenyl phosphate substrate then was added to the wells and allowed to develop for 20C30 min at space temperature. Optical denseness measurements were taken at 405 nm. Mouse Immunizations and Challenge Experiments. All animal procedures were performed in accordance with Institutional Animal Care and Use Committee (IACUC) recommendations and have been authorized by the IACUC of Mount Sinai School of Medicine. BALB/C mice (6- to 8-wk-old) (Jackson Laboratories) were immunized with 25 g LAH-KLH, HA2, KLH only, or PBS in Total Freund’s adjuvant (Sigma) by s.c. administration. Three weeks after main immunization, mice were boosted with 25 g of the same immunogen or with PBS in Incomplete Freund’s adjuvant. Two to three weeks after the boost, mice were challenged with Rabbit Polyclonal to SLC15A1 computer virus. Before computer virus infection, mice were anesthetized Menaquinone-7 by i.p. administration of a mixture of ketamine (75 mg/kg body weight)/xylazine (15 mg/kg body weight). Computer virus was given intranasally in 50 L total PBS; challenge doses consisted of 4 105 pfu X31 or 500 pfu PR8 or HAlo computer virus. Body weights were monitored daily. For passive transfer experiments, mice were bled 2 wk after the last immunization with KLH or LAH-KLH or 3 wk after illness with PR8 computer virus or A/Hong Kong/1/1968 computer virus. Sera from mice were pooled relating to vaccination antigen.