Antibody-independent protection against rotavirus infection of mice stimulated by intranasal immunization with chimeric VP4 or VP6 protein

Abstract

This study was to determine whether individual rotavirus capsid proteins could stimulate protection against rotavirus shedding in an adult mouse model. BALB/c mice were intranasally or intramuscularly administered purified Escherichia coli-expressed murine rotavirus strain EDIM VP4, VP6, or truncated VP7 (TrVP7) protein fused to the 42.7-kDa maltose-binding protein (MBP). One month after the last immunization, mice were challenged with EDIM and shedding of rotavirus antigen was measured. When three 9-microg doses of one of the three rotavirus proteins fused to MBP were administered intramuscularly with the saponin adjuvant QS-21, serum rotavirus immunoglobulin G (IgG) was induced by each protein. Following EDIM challenge, shedding was significantly (P = 0.02) reduced (i.e., 38%) in MBP::VP6-immunized mice only. Three 9-micrograms doses of chimeric MBP::VP6 or MBP::TrVP7 administered intranasally with attenuated E. coli heat-labile toxin LT(R192G) also induced serum rotavirus IgG, but MBP::VP4 immunization stimulated no detectable rotavirus antibody. No protection against EDIM shedding was observed in the MBP::TrVP7-immunized mice. However, shedding was reduced 93 to 100% following MBP::VP6 inoculation and 56% following MBP::VP4 immunization relative to that of controls (P = <0.001). Substitution of cholera toxin for LT(R192G) as the adjuvant, reduction of the number of doses to 1, and challenge of the mice 3 months after the last immunization did not reduce the level of protection stimulated by intranasal administration of MBP::VP6. When MBP::VP6 was administered intranasally to B-cell-deficient microMt mice that made no rotavirus antibody, shedding was still reduced to <1% of that of controls. These results show that mice can be protected against rotavirus shedding by intranasal administration of individual rotavirus proteins and that this protection can occur independently of rotavirus antibody.

FIG. 1

Amylose resin affinity chromatography of chimeric MBP::VP4, MBP::VP6, and MBP::TrVP7. E. coli cells transformed with recombinant plasmids expressing MBP::VP4, MBP::VP6, and MBP::TrVP7 were induced with 0.3 mM IPTG for 3 h. Cell lysates and affinity chromatography-purified chimeric proteins were obtained as described in Materials and Methods. Pure MBP, cell lysates, and purified chimeric proteins were subjected to SDS-PAGE and then electrotransferred to nitrocellulose for Western blot analyses. The blots were incubated with a polyclonal rabbit anti-MBP antiserum. Goat anti-rabbit IgG conjugated to alkaline phosphatase was used as the secondary antibody. BCIP and NBT were used as enzyme substrates to visualize the antibodies bound to chimeric proteins. The arrows indicate putative full-length chimeric MBP::VP4, MBP::VP6, and MBP::TrVP7. Molecular mass markers (in kilodaltons) are noted at the left.

FIG. 2

Western blot analyses of sera from mice immunized with chimeric rotavirus proteins. CsCl-purified TL rotavirus particles were subjected to SDS-PAGE and then electrotransferred onto a nitrocellulose sheet, which was then cut into strips for Western blot analyses. The strips were incubated with sera from mice which had been immunized i.n. with MBP::VP4, MBP::VP6, or MBP::TrVP7, each in combination with LT(R192G) (A), or i.m. with the same proteins in combination with QS-21 (B). Goat anti-mouse IgG conjugated to alkaline-phosphatase was used as the secondary antibody. BCIP and NBT were used to visualize antibodies bound to VP4, VP6, or VP7.

FIG. 3

Protection from shedding of rotavirus antigen in mice inoculated with chimeric subunit vaccines. BALB/c mice were inoculated either i.n. or i.m. with three 9-μg doses of either MBP::VP4, MBP::VP6, or MBP::TrVP7 with or without an adjuvant. The adjuvants used for i.n. and i.m. inoculations were LT(R192G) (10 μg) and QS-21 (20 μg), respectively. One month after the last immunization, mice were challenged by oral gavage with 4 × 10⁴ FFU of passage 9 EDIM. Stool samples were collected on the day of challenge and for the subsequent 7 days. Quantities of rotavirus antigen shed were determined by ELISA. The total quantity shed by each group was compared with the total quantity shed by the control group (MBP and adjuvant) and expressed as percent reduction in shedding relative to the level of shedding of the control group. The * symbols indicate that levels of shedding by immunized groups were significantly lower (P ≤0.02) than the level of shedding of uninoculated mice.

FIG. 4

Protection from shedding of rotavirus antigen in mice immunized with chimeric VP6. BALB/c and μMt mice were inoculated i.n. with two 9-μg doses of MBP::VP6 and LT(R192G). One month after the last immunization, mice were challenged by oral gavage with 4 × 10⁴ FFU of passage 9 (BALB/c) or 5 × 10⁵ FFU of unpassaged EDIM (μMt). Stool samples were collected on the day of challenge and for 7 days thereafter. Quantities of rotavirus antigen shed each day were determined by ELISA. The numbers in the graphs are quantities of rotavirus antigen shed by immunized mice.