Intranasal delivery of a bivalent norovirus vaccine formulated in an in situ gelling dry powder

Abstract

The global health community is beginning to understand the burden of norovirus-associated disease, which has a significant impact in both developed and developing countries. Norovirus virus like particle (VLP)-based vaccines are currently under development and have been shown to elicit systemic and mucosal immune responses when delivered intranasally. In the present study, we describe the use of a dry powder formulation (GelVac™) with an in situ gelling polysaccharide (GelSite™) extracted from Aloe vera for nasal delivery of a bivalent vaccine formulation containing both GI and GII.4 norovirus VLPs. Dose-ranging studies were performed to identify the optimal antigen dosages based on systemic and mucosal immune responses in guinea pigs and determine any antigenic interference. A dose-dependent increase in systemic and mucosal immunogenicity against each of the VLPs were observed as well as a boosting effect for each VLP after the second dosing. A total antigen dose of ≥50 μg of each GI and GII.4 VLPs was determined to be the maximally immunogenic dose in guinea pigs. The immunogenicity results of this bivalent formulation, taken together with previous work on monovalent GelVac™ norovirus vaccine formulation, provides a basis for future development of this norovirus VLP vaccine.

Fig 1. Transmission electron microscopy of norovirus VLPs.

GI (A) and GII.4 (B) VLPs were dissolved in water and imaged at 150,000x magnification (scale bar 100 μm). VLP particles were spherical in appearance at the expected size of 23–38 nm.

Fig 2. Serum norovirus-specific IgG and IgA production following intranasal immunization with GelVac^™ bivalent and monovalent vaccine powders.

Female Hartley guinea pigs were immunized intranasally with 20 mg of a bivalent vaccine powder formulation containing various amounts of GI and GII.4 VLPs on days 0 and 21. Serum samples were collected on day 0, 14, 21, 42, and 56 and analyzed for specific IgG antibodies against GI (A) and GII.4 (B). Serum samples were also analyzed for specific IgA antibodies against GI (C) and GII.4 (D). Error bars are provided as geometric standard deviation. *p<0.05 as compared to the placebo control group. Horizontal dotted line depicts the limit of detection for these assays.

Fig 3. Neutralizing antibody production following intranasal immunization with GelVac^™ dry powder bivalent and monovalent vaccine.

Female Hartley guinea pigs were immunized intranasally with 20 mg of a bivalent vaccine powder formulation containing various amounts of GI and GII.4 VLPs on days 0 and 21. Serum samples were collected on days 0, 14, 21, 42, and 56 and analyzed for GI (A) and GII.4 (B) neutralizing antibodies. Error bars are provided as geometric standard deviation. *p<0.05 as compared to the placebo control group. Horizontal dotted line depicts the limit of detection for these assays.

Fig 4. Mucosal norovirus-specific antibody production following intranasal immunization with GelVac^™ dry powder bivalent and monovalent vaccine.

Female Hartley guinea pigs were immunized intranasally with 20 mg of a bivalent vaccine powder formulation containing various amounts of GI and GII.4 VLPs on days 0 and 21. Vaginal lavage samples were collected on days 0, 14, 21, 42, and 56 and analyzed for GI (A) and GII.4 (B) norovirus-specific antibodies. On day 56, animals were euthanized and intestinal lavage samples were analyzed for GI (C) and GII.4 (D) norovirus-specific antibodies. Error bars are provided as geometric standard deviation. *p<0.05 as compared to the placebo control group. Horizontal dotted line depicts the limit of detection for these assays.