Heterotypic protection and induction of a broad heterotypic neutralization response by rotavirus-like particles

Abstract

The recognition that rotaviruses are the major cause of life-threatening diarrheal disease and significant morbidity in young children has focused efforts on disease prevention and control of these viruses. Although the correlates of protection in children remain unclear, some studies indicate that serotype-specific antibody is important. Based on this premise, current live attenuated reassortant rotavirus vaccines include the four predominant serotypes of virus. We are evaluating subunit rotavirus vaccines, 2/6/7-VLPs and 2/4/6/7-VLPs, that contain only a single VP7 of serotype G1 or G3. In mice immunized parenterally twice, G3 virus-like particles (VLPs) induced a homotypic, whereas G1 VLPs induced a homotypic and heterotypic (G3) serum neutralizing immune response. Administration of three doses of G1 or G3 VLPs induced serum antibodies that neutralized five of seven different serotype test viruses. The inclusion of VP4 in the VLPs was not essential for the induction of heterotypic neutralizing antibody in mice. To confirm these results in another species, rabbits were immunized parenterally with two doses of 2/4/6/7-VLPs containing a G3 or G1 VP7, sequentially with G3 VLPs followed by G1 (G3/G1) VLPs, or with live or psoralen-inactivated SA11. High-titer homotypic serum neutralizing antibody was induced in all rabbits, and low-level heterotypic neutralizing antibody was induced in a subset of rabbits. The rabbits immunized with the G1 or G3/G1 VLPs in QS-21 were challenged orally with live G3 ALA rotavirus. Protection levels were similar in rabbits immunized with homotypic G3 2/4/6/7-VLPs, heterotypic G1 2/4/6/7-VLPs, or G3/G1 2/4/6/7-VLPs. Therefore, G1 2/4/6/7-VLPs can induce protective immunity against a live heterotypic rotavirus challenge in an adjuvant with potential use in humans. Following challenge, broad serum heterotypic neutralizing antibody responses were detected in rabbits parenterally immunized with G1, G3/G1, or G3 VLPs but not with SA11. Immunization with VLPs may provide sufficient priming of the immune system to induce protective anamnestic heterotypic neutralizing antibody responses upon subsequent rotavirus infection. Therefore, a limited number of serotypes of VLPs may be sufficient to provide a broadly protective subunit vaccine.

FIG. 1

SDS-PAGE and Western blot analysis of VLP preparations. The proteins in purified SA11 (lane 1) or VLPs (G3 2/6/7-VLPs [lane 2], G3 2/4/6/7-VLPs [lane 3]), G1 2/6/7-VLPs [lane 4], and G1 2/4/6/7-VLPs [lane 5]), made in infected insect cells and purified, were separated by SDS-PAGE, transferred to nitrocellulose, and detected with a hyperimmune anti-SA11 mouse serum (A) or an anti-VP4 monoclonal antibody, 5E4 (B). Locations of the individual proteins are shown at the left and right.

FIG. 2

G1 VLPs induce heterotypic neutralizing antibody in mice. Groups of five CD-1 mice were parenterally immunized with the indicated VLPs in Freund’s adjuvant. The serum (28 dpi) neutralizing antibody titers against SA11(P5B,G3) (white bars) and Wa (P1A,G1) (solid bars) were assayed by PRN; the geometric mean of the titers are compared. Statistical differences (P < 0.05) in GMTs between groups are indicated by the same symbol (■, ⧫, or □). The GMT against Wa for the G3 VLPs are statistically different from all other groups and are not denoted by a symbol. The error bars represent 1 standard error of the mean.

FIG. 3

Serum antibody responses in mice immunized parenterally with the indicated VLPs in Freund’s adjuvant. The serum antirotavirus antibody titers were assayed by ELISA (five mice per group); the GMTs determined for mice twice immunized 28 dpi (solid bar) and 42 dpi (hatched bar) or three times immunized 114 dpi (white bar) are compared. Statistical increases (P < 0.05) in GMTs in each of the groups are indicated by the same symbol (■ or ⧫). The error bars represent 1 standard error of the mean.

FIG. 4

Serum and fecal antibody responses in rabbits immunized twice parenterally with buffer (mock), SA11, G3 and G1 2/4/6/7-VLPs sequentially, or G1 2/4/6/7-VLPs and challenged orally with ALA (G3). Prechallenge (■) and postchallenge (□) serum and fecal samples were assayed by ELISA for total serum antirotavirus antibody (A), rotavirus-specific IgG fecal antibody (B), or rotavirus-specific IgA fecal antibody (C). The error bars represent 1 standard error of the mean. The GMTs of the rabbits immunized sequentially with the G3 and G1 or the G1 2/4/6/7-VLPs are compared to the GMTs for mock- and SA11-immunized animals as previously reported (7). Significant increases in GMT postchallenge are indicated by * (P < 0.05).