Immune mechanisms responsible for vaccination against and clearance of mucosal and lymphatic norovirus infection

Abstract

Two cardinal manifestations of viral immunity are efficient clearance of acute infection and the capacity to vaccinate against secondary viral exposure. For noroviruses, the contributions of T cells to viral clearance and vaccination have not been elucidated. We report here that both CD4 and CD8 T cells are required for efficient clearance of primary murine norovirus (MNV) infection from the intestine and intestinal lymph nodes. Further, long-lasting protective immunity was generated by oral live virus vaccination. Systemic vaccination with the MNV capsid protein also effectively protected against mucosal challenge, while vaccination with the capsid protein of the distantly related human Lordsdale virus provided partial protection. Fully effective vaccination required a broad immune response including CD4 T cells, CD8 T cells, and B cells, but the importance of specific immune cell types varied between the intestine and intestinal lymph nodes. Perforin, but not interferon gamma, was required for clearance of MNV infection by adoptively transferred T lymphocytes from vaccinated hosts. These studies prove the feasibility of both mucosal and systemic vaccination against mucosal norovirus infection, demonstrate tissue specificity of norovirus immune cells, and indicate that efficient vaccination strategies should induce potent CD4 and CD8 T cell responses.

Figure 1. Short-term vaccination against MNV using live MNV strains and VRPs expressing ORF1, ORF2, and ORF3 from MNV and ORF2 from Chiba virus and Lordsdale virus.

(A) Vaccination protocol used in short-term vaccination. Viral titers in (B) distal ileum and (C) MLN of adult (8-week-old) WT mice after MNV1.CW3 challenge following vaccination with the indicated vaccines. Viral titers in (D) distal ileum and (E) MLN of adult (8-week-old) or aged (14-month-old) WT mice immunized with MNV1.CW3 ORF2 VRP or HA VRP and challenged with MNV1.CW3. These data are pooled from two independent experiments with 3-5 mice per group in each experiment. (**) indicates p<0.0001, (*) indicates p<0.05. Unless otherwise stated, live virus vaccinations are compared to vaccination with reovirus, and VRP vaccinations are compared to HA VRP. LD indicates the limit of detection. Bars indicate the arithmetic mean.

Figure 2. Long-term vaccination against MNV using a live MNV strain and ORF2 VRPs.

(A) Vaccination protocol used in long-term vaccination. Viral titers in (B) distal ileum and (C) MLN of WT mice after MNV1.CW3 challenge following vaccination with indicated vaccines. These data are pooled from three independent experiments with 3–5 mice per group in each experiment. (**) indicates p<0.0001, (*) indicates p<0.05. Unless otherwise stated, live virus vaccinations are compared to vaccination with reovirus, and VRP vaccinations are compared to HA VRP. LD indicates the limit of detection.

Figure 3. Complete short-term protection against MNV infection requires MHC Class II, MHC Class I, β2M, and B cells.

(A) Vaccination protocol used in short-term vaccination using immunodeficient mice. Viral titers in (B) distal ileum, (C) MLN of B cell-/, MHC Class II-/-, and MHC Class I×β2M-/- mice after MNV1.CW3 challenge following short-term vaccination with the indicated vaccines. These data are pooled from three independent experiments with 3–5 mice per group in each experiment. (**) indicates p<0.0001, (*) indicates p<0.05. Unless otherwise stated, live virus vaccinations are compared to vaccination with reovirus, and VRP vaccinations are compared to HA VRP. LD indicates the limit of detection.

Figure 4. MHC Class II limits early MNV replication, and deficiency in MHC Class II or MHC Class I & β2M delays MNV clearance.

(A) Protocol of challenge used in experiments in this figure. Viral titers in distal ileum (B,D) and MLN (C,E) of WT and MHC Class I×β2M-/- mice (B,C) and MHC Class II-/- mice (D,E) after infection with MNV1.CW3. These data are pooled from two to three independent experiments with 3–5 mice per group in each experiment. (**) indicates p<0.0001, (*) indicates p<0.05. LD indicates the limit of detection.

Figure 5. CD4 and CD8 T cells are required for clearance of primary MNV infection at 6 days post-infection.

(A) Representative flow cytometric analysis of CD4 and CD8 staining on splenocytes harvested from control and immunodepleted WT mice 6 days post-infection with MNV1.CW3. Viral titer in (B) distal ileum and (C) MLN after treatment with the indicated antibodies. Results are pooled from two independent experiments with 3–5 mice per group in each experiment. (**) indicates p<0.0001, (*) indicates p<0.05. LD indicates the limit of detection.

Figure 6. RAG1-/- mice fail to clear MNV infection in the intestine.

Viral titers in (A) duodenum/jejunum and (B) distal ileum of RAG1-/- mice after infection with MNV1.CW3. Results are pooled from four independent experiments with 5 mice per group in each experiment. LD indicates the limit of detection.

Figure 7. Immune CD4 and CD8 T cells are both required, and perforin plays a role in clearance of persistent MNV infection in RAG1-/- recipients.

(A) Representative flow cytometric analysis of splenocytes harvested from RAG1-/- recipient mice 6 days post-transfer of splenocytes. Viral titers in (B) duodenum/jejunum and (C) distal ileum 6 days after adoptive transfer of medium alone, WT non-immune splenocytes (N–I), WT immune splenocytes (WT), WT immune splenocytes with or without depleting antibodies or immune splenocytes from IFNγ-/- or perforin-/- (Pfn-/-) mice. These data are pooled from three independent experiments with 3–5 mice per group in each experiment. (**) indicates p<0.0001, (*) indicates p<0.05. LD indicates the limit of detection.