Parenterally Administered Norovirus GII.4 Virus-Like Particle Vaccine Formulated with Aluminum Hydroxide or Monophosphoryl Lipid A Adjuvants Induces Systemic but Not Mucosal Immune Responses in Mice

Abstract

Norovirus (NoV) is a main cause of acute gastroenteritis across all ages worldwide. NoV vaccine candidates currently in clinical trials are based on noninfectious highly immunogenic virus-like particles (VLPs) delivered intramuscularly (IM). Since NoV is an enteric pathogen, it is likely that mucosal immunity has a significant role in protection from infection in the intestine. Due to the fact that IM delivery of NoV VLPs does not generate mucosal immunity, we investigated whether NoV genotype GII.4 VLPs coadministered with aluminum hydroxide (Al(OH)₃) or monophosphoryl lipid A (MPLA) would induce mucosal antibodies in mice. Systemic as well as mucosal IgG and IgA antibodies in serum and intestinal and nasal secretions were measured. As expected, strong serum IgG, IgG1, and IgG2a antibodies as well as a dose sparing effect were induced by both Al(OH)₃ and MPLA, but no mucosal IgA antibodies were detected. In contrast, IN immunization with GII.4 VLPs without an adjuvant induced systemic as well as mucosal IgA antibody response. These results indicate that mucosal delivery of NoV VLPs is needed for induction of mucosal responses.

Figure 1

NoV GII.4-specific systemic IgG (a, b) and IgA (c, d) antibody responses after IM immunization with 0.3 or 10 μg of NoV GII.4 VLPs alone or 0.3 μg of VLPs combined with Al(OH)₃ or MPLA, or IN immunization with 10 μg of VLPs alone. Group mean OD₄₉₀ values with standard error of the means of IgG (a) and IgA (c) antibodies in 1 : 200 (a) and 1 : 20 (c) diluted sera of experimental mice. Control mice received PBS only. End-point titers of IgG (b) and IgA (d) antibodies in sera of the experimental groups. Bars represent reciprocal of log₁₀ geometric mean titers with 95% confidence intervals. The number of positive/tested mice is denoted on each figure in parenthesis.

Figure 2

Development of IgG (a) and IgG subtype antibodies (b, c) in mice immunized IM with 0.3 μg of GII.4 VLPs formulated with Al(OH)₃ or MPLA or 10 μg of GII.4 VLPs alone. (a) Kinetics of NoV GII.4-specific total IgG antibodies in sera of mice immunized with the antigenic formulations at study weeks 0 and 3. Control mice received PBS only. Group mean OD₄₉₀ values with standard error of the means of individual tail blood samples collected at indicated study weeks and termination sera at week 5 are shown. End-point titrations of anti-GII.4 IgG1 (b) and IgG2a (c) antibodies in termination sera at week 5. Mean titration curves with standard errors of the mean of the experimental groups are shown.

Figure 3

NoV GII.4-specific fecal antibody responses after IM immunization with 0.3 or 10 μg of NoV GII.4 VLPs alone or 0.3 μg of VLPs combined with Al(OH)₃ or MPLA, or IN immunization with 10 μg of VLPs alone. End-point titrations of anti-GII.4 IgG (a) and IgA (b) antibodies in 10% fecal suspensions of experimental mice. Control mice received PBS only. Mean titration curves with standard errors of the mean of the experimental groups are shown.

Figure 4

Mucosal antibody responses in nasal washes after IM immunization with 0.3 μg of NoV GII.4 VLPs formulated with Al(OH)₃ or MPLA, or IM or IN immunization with 10 μg of VLPs alone. End-point titrations of anti-GII.4 IgG (a) and IgA (b) antibodies in nasal washes (NWs) of experimental mice. Control mice received PBS only. Mean titration curves with standard errors of the mean of the experimental groups are shown.

Figure 5

IgG and IgA antibodies in cells from mesenteric lymph nodes after IM immunization with 0.3 μg of NoV GII.4 VLPs formulated with Al(OH)₃ (a) or MPLA (b). End-point titration curves of anti-GII.4 IgG and IgA antibodies in cell culture supernatants from pooled mesenteric lymph nodes (MLNs) of experimental groups stimulated in vitro with 5 μg GII.4 VLPs. Control mice received PBS only.