An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles

Abstract

Virus-like particles (VLPs) derived from enteric pathogens like Norwalk virus (NV) are well suited to study oral immunization. We previously described stable transgenic plants that accumulate recombinant NV-like particles (rNVs) that were orally immunogenic in mice and humans. The transgenic approach suffers from long generation time and modest level of antigen accumulation. We now overcome these constraints with an efficient tobacco mosaic virus (TMV)-derived transient expression system using leaves of Nicotiana benthamiana. We produced properly assembled rNV at 0.8 mg/g leaf 12 days post-infection (dpi). Oral immunization of CD1 mice with 100 or 250 microg/dose of partially purified rNV elicited systemic and mucosal immune responses. We conclude that the plant viral transient expression system provides a robust research tool to generate abundant quantities of rNV as enriched, concentrated VLP preparations that are orally immunogenic.

Fig. 1

3′ ICON modules used in the study. LB and RB, left and right borders of the T-DNA region; AttB, specific recombination site from phage PhiC31; I, intron; NTR, 3′ non translated region; Nos-T, nopaline synthase terminator.

Fig. 2

Western blot analysis of the NVCP produced in N. benthamiana leaves harvested at 12 dpi. The membrane was probed with rabbit anti-NVCP serum. Lane 1, insect cell-derived NVCP reference standard; lane 2, uninfected leaf extract; lane 3, extract from pICH-GFP infiltrated leaves; 4, crude extract from pICH-sNVCP infiltrated leaves, lane 5, rNV partially purified from pICH-sNVCP infiltrated leaves.

Fig. 3

Evaluation of rNV particle formation by sucrose gradient sedimentation. Insect cell-derived rNV (i-rNV) and crude extracts from N. benthamiana infiltrated with pICH-sNVCP (cp-rNV) were sedimented on a 10-60% sucrose gradient; 15 fractions were collected. The distribution of NVCP across gradient fractions was determined by ELISA for NVCP. The top of the gradient is at left.

Fig. 4

Visualization by negative staining and electron microscopy of rNV. (A) insect cell-derived rNV; (B) rNV partially purified by sucrose gradient from pICH-sNVCP infected N. benthamiana leaves.

Fig. 5

Partial purification of plant derived rNV. (A) Protein samples were analyzed on a 4-20% gradient polyacrylamide gel and visualized by Coomassie staining. Lane 1, Molecular weight marker; lane 2, insect cell-derived rNV reference standard (2 μg); lane 3, crude protein extract from uninfected leaves (20 μg); lane 4, crude protein extract from pICH-sNVCP infiltrated leaves (20 μg); lane 5, rNV partially purified from pICH-sNVCP infected N. benthamiana leaves (20 μg). NVCP, Norwalk virus capsid protein; LSR, large subunit of Rubisco; SSR, small subunit of Rubisco. (B) ELISA profile of sucrose gradient fractions of rNV. i-rNV, insect cell derived rNV; pp-rNV, partially purified extracts from N. benthamiana leaves infiltrated with pICH-sNVCP. The top of the gradient is at left. (C) Western blot analysis of sucrose gradient fractions probed with rabbit polyclonal anti-NVCP.

Fig. 6

Time course of NVCP specific serum IgG (A) and vaginal IgA (B) responses in mice after oral delivery of rNV produced in N. benthamiana leaves. CD1 mice (n=7 per group) were gavaged on weeks 0, 3 and 6, as indicated by arrows (↑). Group 1 received 100 μg rNV; group 2, 250 μg rNV; group 3, 100 μg rNV + 10 μg CT; and group 4, 250 μg rNV + 10 μg CT. Serum IgG and vaginal IgA were measured by ELISA. The y-axis shows the geometric means titers (GMT) and the error bars show the standard error of the mean.

Fig. 7

Intestinal IgA responses in CD1 mice to different doses of partially purified plant-derived rNV administered by gavage in presence or absence of CT. The fecal rNV-specific IgA titer was evaluated at 5 and 8 weeks post oral immunization and expressed as GMT. The error bars show the standard error of the mean.