Generation and characterization of a trackable plant-made influenza H5 virus-like particle (VLP) containing enhanced green fluorescent protein (eGFP).

Medicago, Inc. has developed an efficient virus-like particle (VLP) vaccine production platform using the Nicotiana benthamiana expression system, and currently has influenza-based products targeting seasonal/pandemic hemagglutinin (HA) proteins in advanced clinical trials. We wished to generate a trackable HA-based VLP that would allow us to study both particle assembly in plants and VLP interactions within the mammalian immune system. To this end, a fusion protein was designed, composed of H5 (from influenza A/Indonesia/05/2005 [H5N1]) with enhanced green fluorescent protein (eGFP). Expression of H5-eGFP in N. benthamiana produced brightly fluorescent ∼160 nm particles resembling H5-VLPs. H5-eGFP-VLPs elicited anti-H5 serologic responses in mice comparable to those elicited by H5-VLPs in almost all assays tested (hemagglutination inhibition/IgG(total)/IgG1/IgG2b/IgG2a:IgG1 ratio), as well as a superior anti-GFP IgG response (mean optical density = 2.52 ± 0.16 sem) to that elicited by soluble GFP (mean optical density = 0.12 ± 0.06 sem). Confocal imaging of N. benthamiana cells expressing H5-eGFP displayed large fluorescent accumulations at the cell periphery, and draining lymph nodes from mice given H5-eGFP-VLPs via footpad injection demonstrated bright fluorescence shortly after administration (10 min), providing proof of concept that the H5-eGFP-protein/VLPs could be used to monitor both VLP assembly and immune trafficking. Given these findings, this novel fluorescent reagent will be a powerful tool to gain further fundamental insight into the biology of influenza VLP vaccines.

Immunologic characterization of a novel inactivated nasal mumps virus vaccine adjuvanted with Protollin.

An inactivated, mucosal mumps virus (MuV) vaccine would address many of the problems associated with current live-attenuated formulations. Protollin (Prl)-based adjuvants (containing TLR2 and TLR4 ligands) are well-suited for nasal administration. We sought to develop an inactivated whole-virus nasal vaccine for MuV using the Prl adjuvant/delivery vehicle and to test tolerability and immunogenicity in a mouse model. BALB/c mice exhibited signs of transient reactogenicity (hunched posture, erect fur, weight loss ≤10% of total body weight) following administration of intranasal MuV-Prl vaccines, though most of these manifestations resolved within 24h. Compared to high-dose unadjuvanted vaccine (8µgMuV), administration of high-dose adjuvanted formulation (8µgMuV-Prl) induced greater MuV-specific serum IgG (3.26E6ng/mL vs. 2.2E5ng/mL, 8µgMuV-Prl vs. 8µgMuV, p<0.001) and mucosal IgA (128ng/mL vs. 45ng/mL, 8µgMuV-Prl vs. 8µgMuV, p<0.05). Serum IgG isotypes and splenocyte cytokine secretion induced by MuV-Prl suggested a predominant T helper cell (Th)1-type immune response. This response was characterized by: (1) ≥four-fold increase of IgG2a levels compared to IgG1; and (2) high IL-2 (644pg/mL)/IFN-γ (228pg/mL) and low IL-5 (31pg/mL) secretion in MuV-restimulated splenocytes from animals receiving MuV-Prl formulations. MuV-Prl vaccination induced higher levels of serum antibodies capable of neutralizing MuV in vitro than MuV alone, particularly for high-dose 8µg formulations (357 neutralizing units (NU)/mL vs. 32NU/mL, 8µgMuV-Prl vs. 8µgMuV, p<0.001). Thus, nasal MuV-Prl vaccines are fairly well-tolerated and highly immunogenic in mice.