CD4 T cells are the only lymphocytes needed to protect mice against rotavirus shedding after intranasal immunization with a chimeric VP6 protein and the adjuvant LT(R192G)

Abstract

Intranasal immunization of mice with a chimeric VP6 protein and the mucosal adjuvant Escherichia coli heat labile toxin LT(R192G) induces nearly complete protection against murine rotavirus (strain EDIM [epizootic diarrhea of infant mice virus]) shedding for at least 1 year. The aim of this study was to identify the protective lymphocytes elicited by this new vaccine candidate. Immunization of mouse strains lacking one or more lymphocyte populations revealed that protection was dependent on alphabeta T cells but mice lacking gammadelta T cells and B cells remained fully protected. Furthermore, depletion of CD8 T cells in immunized B-cell-deficient mice before challenge resulted in no loss of protection, while depletion of CD4 T cells caused complete loss of protection. Therefore, alphabeta CD4 T cells appeared to be the only lymphocytes required for protection. As confirmation, purified splenic T cells from immunized mice were intraperitoneally injected into Rag-2 mice chronically infected with EDIM. Transfer of 2 x 10(6) CD8 T cells had no effect on shedding, while transfer of 2 x 10(5) CD4 T cells fully resolved shedding in 7 days. Interestingly, transfer of naive splenic CD4 T cells also resolved shedding but more time and cells were required. Together, these results establish CD4 T cells as effectors of protection against rotavirus after intranasal immunization of mice with VP6 and LT(R192G).

FIG. 1.

Effects of i.n. immunization with VP6 and LT(R192G) on protection against shedding of rotavirus antigen in BALB/c (A) or C57BL/6 (B) mice after EDIM challenge. Groups of eight mice were either not immunized or immunized with two doses of antigen and adjuvant (separated by 2 weeks) and challenged with 1,000 SD₅₀ of wild-type EDIM 4 weeks after the second dose. Stools were collected daily and analyzed by ELISA to determine quantities of rotavirus antigen. Error bars represent standard deviations.

FIG. 2.

Effects of either CD8 or CD4 T-cell depletion on shedding of rotavirus antigen in either naive or VP6-immunized, B-cell-deficient J_HD mice during the 7 days after EDIM challenge. Groups of six J_HD mice were either not immunized or i.n. immunized with two doses of MBP::VP6 and LT(R192G) separated by 2 weeks. Starting at 24 days after the second dose, some groups of mice were depleted of either CD8 or CD4 T cells by daily (4 consecutive days) injections with MAbs specific for each cell type. On day 28 after the second dose, all mice were challenged with 1,000 SD₅₀ of wild-type EDIM and monitored daily for shedding of rotavirus antigen during the following 7 days. Two additional MAb injections were administered during the 7-day analysis period. The results represent the average amounts in nanograms (ng) of rotavirus antigen shed/mouse/day during the 7-day period, with standard deviations shown by the error bars.

FIG. 3.

Rotavirus shedding in Rag-2 mice after adoptive transfer of purified CD4 T cells from VP6-immunized mice. A group of four BALB/c mice were immunized i.n. with two doses of MBP::VP6 and LT(R192G) and 4 weeks after the second dose were sacrificed. Their splenic CD4 T cells were column purified and sorted twice prior to i.p. injection of different quantities into groups of eight Rag-2 mice that were chronically shedding large quantities of rotavirus after oral inoculation of 1,000 SD₅₀ of wild-type EDIM 26 days earlier (day 0). EDIM shedding continued to be monitored daily in all groups until day 60. Starting on day 61, the two groups that received the larger numbers of CD4 T cells were depleted with anti-CD4 MAb injections for 4 consecutive days, and shedding of rotavirus antigen continued to be monitored until day 80.

FIG. 4.

FACS analysis of either B cells or CD8 or CD4 T cells obtained from the spleens, IELs, or lamina propria of Rag-2 mice 7 weeks after adoptive transfer of purified splenic CD4 T cells from VP6-immunized mice. Splenic CD4 T cells were obtained 4 weeks after the second i.n. immunization of BALB/c mice with MBP::VP6 and LT(R192G), column purified and sorted twice. These cells (2 × 10⁵) were injected i.p. into Rag-2 mice that were chronically shedding large amounts of EDIM. At 7 weeks after transfer, a group of four Rag-2 mice were sacrificed and their splenic and intestinal cells were collected, stained with MAbs against the cell markers specified, and analyzed by FACS (26).

FIG. 5.

Rotavirus shedding in Rag-2 mice after adoptive transfer of purified CD4 T cells from naive mice. A group of five naive BALB/c mice were sacrificed, and their splenic CD4 T cells were column purified and sorted twice. Specified quantities of these cells were then injected (i.p.) into groups of four Rag-2 mice that were chronically shedding high levels of rotavirus after oral inoculation of 1,000 SD₅₀ of wild-type EDIM 26 days earlier (day 0). Monitoring for rotavirus antigen shedding was continued until day 61.