was a full-time employee of Protein Sciences Corp.; and S.G.R. future outbreaks of lethal pandemic flu. H5N1 is a highly pathogenic avian influenza virus that can cause severe disease and death in humans, and world health authorities agree that the potential for pandemic H5N1 infection is high. Vaccination remains the most effective mechanism for preventing influenza, but there are complex challenges in implementing a pandemic preparedness plan, including: an inability to rapidly deploy the vast numbers of safe and effective doses needed on a worldwide scale; the fact that the immunogenicity of current nonadjuvanted H5N1 vaccines are relatively weak and require large antigen doses; and the potency of stockpiled prepandemic vaccines may be severely limited given the anticipated antigenic drift/shift associated with the emergence of a novel strain of pandemic H5N1. The US government has outlined provisions for new technologies that maximize immunogenicity and manufacturing capacity of vaccines for influenza, including the use of recombinant protein-based vaccines and adjuvants, which augment immunity and dose-sparing capacity. The most advanced egg-free flu vaccine candidate is a recombinant multimeric H5 hemagglutinin protein (rH5) produced by using a baculovirus expression vector system in SF+ insect cells (1, 2). Previous clinical studies suggested that two 90-g doses of rH5 induced modest responses equivalent to conventional subvirion-based H5N1 vaccines (3, 4). This finding has prompted efforts to test rH5 with an adjuvant. Currently, the leading H5N1 vaccine adjuvants are oil-in-water (o/w) emulsions, which augment neutralizing antibody titers, increase the breadth of cross-reactive antibodies, and possess significant dose-sparing activity (5, 6). Importantly, these adjuvants are particularly effective in priming na?ve individuals in the absence of preexisting memory. Vaccine adjuvants regulate adaptive immunity by stimulating dendritic cell maturation and antigen presentation (7, 8). A leading adjuvant target on DC is the family of ML213 innate Toll-like receptors, particularly the LPS receptor, Toll-like receptor 4 (TLR4). Glucopyranosyl lipid adjuvant (GLA) is a formulated form of the synthetic TLR4 agonist PHAD (Avanti Polar Lipids), which is analogous to the detoxified LPS derivative monophosphoryl lipid A (MPL), a component of the human papillomavirus vaccine Cervarix (9). Experimental vaccines containing GLA demonstrate enhanced immunogenicity in a variety of disease models (8), and ML213 in the context of influenza, GLA formulated in a stable emulsion (GLA-SE) improved Fluzone-dependent antibody titers in mice and nonhuman ML213 primates, relative to an emulsion alone (10C13). Given the critical importance of immunological priming for pandemic vaccine preparedness, we set out to test whether adjuvanting a recombinant H5 antigen with GLA-SE would broaden protective immunity against H5N1. Results We have established an assay for adjuvant-dependent priming of a protective immune response. The kinetics of this response were measured by immunizing BALB/c mice once with 50 ng of rH5 derived from A/Vietnam/1203/04 (rH5VN) protein adjuvanted with GLA-SE or SE and then challenging animals after increasing the number of days with a 100 LD50 (500 pfu) dose of A/Vietnam/1203/04 virus. As indicated in Fig. 1and 0.05; Fishers test). The limit of detection for ELISA is an endpoint titer of 3, and the asterisk denotes a significant difference in endpoint titers ( 0.05; Rabbit Polyclonal to EIF2B3 ANOVA). ( 0.05) between SE and GLA-SE are indicated by the asterisks. The increased IgG2c:IgG1 titer ratio, indicative of a Th1 response, is shown in 0.05). In a separate experiment, C57BL/6 mice (8 per group) were treated with CD4-depleting GK1.5 MAb and control LTF-2 MAb, immunized with adjuvanted rH5VN (200 g), and after virus challenge (1,000 LD50) on d14, assayed for HI activity (and 0.05) between SE and GLA-SE are indicated by the asterisks. (= 0.028; Fishers exact test). Open in a separate window Fig..
