Short-term immunoglobulin A B-cell memory resides in intestinal lymphoid tissues but not in bone marrow of gnotobiotic pigs inoculated with Wa human rotavirus

Abstract

Immunological memory is important for protecting the host from reinfection. To investigate the development and sites of residence of intestinal memory B cells, and their role in protective immunity to reinfection with an enteric virus, we assessed the association between memory B cell and antibody-secreting cell (ASC) responses and protection using a gnotobiotic pig model for human rotavirus (HRV) infection and diarrhoea. The isotypes, quantities and tissue distribution of rotavirus-specific memory B cells and ASC were evaluated prechallenge (28 and 83 postinoculation days [PID]) and postchallenge (7 postchallenge days [PCD]), using enzyme-linked immunospot (ELISPOT) assay, in gnotobiotic pigs inoculated once with virulent or three times with attenuated HRV and challenged at PID 28 with the corresponding virulent HRV. Complete protection against HRV shedding and diarrhoea was associated with significantly higher numbers of immunoglobulin A (IgA) and immunoglobulin G (IgG) memory B cells and ASC in the ileum of virulent HRV-inoculated pigs at challenge. In contrast, pigs inoculated with attenuated HRV had lower numbers of IgA and IgG memory B cells and ASC in intestinal lymphoid tissues, but higher numbers in the spleen. The bone marrow had the lowest mean numbers of IgA and IgG memory B cells and ASC prechallenge in both groups of HRV-inoculated pigs. Therefore, bone marrow was not a site for IgA and IgG rotavirus-specific antibody production or for memory B cells after inoculation with live rotavirus, from 28 PID up to at least 83 PID. The effect of in vitro antigen dose was examined and it was determined to play an important role in the development of ASC from memory B cells for the different tissues examined.

Figure 1

The effect of rotavirus-stimulating antigen dose on the numbers of isotype-specific memory B cells detected by in vitro enzyme-linked immunospot (ELISPOT) assay. Mononuclear cells (MNC) from the ileum, mesenteric lymph nodes (MLN) and spleen of pigs inoculated orally and challenged with live virulent WA human rotavirus (HRV) were extracted on postinoculation days (PID) 21–28 (postchallenge day [PCD] 0). An in vitro ELISPOT assay was performed after stimulation of the MNC with different doses of HRV antigen for 5 days in cell culture. Data represent the mean numbers of Wa HRV-specific memory B cells per 5 × 10⁵ MNC for four pigs.

Figure 2

Isotype-specific antibody-secreting cells (ASC) and memory B cells to Wa human rotavirus (HRV) in gnotobiotic pigs following oral inoculation with live virulent or attenuated Wa HRV. Mononuclear cells (MNC) from the duodenum, ileum, mesenteric lymph nodes (MLN), spleen and bone marrow (BM) of pigs were extracted and assayed on postinoculation day (PID) 28 (postchallenge day [PCD] 0). Enzyme-linked immunospot (ELISPOT) assays for determining in vivo HRV-activated antibody-secreting cell (ASC) numbers were performed on the day of MNC extraction. ELISPOT assays for determining memory B-cell numbers were carried out after the MNC had been stimulated in vitro with HRV antigen in cell culture for 5 days. Data represent the mean numbers of Wa HRV-specific ASC or memory B cells per 5 × 10⁵ MNC for three to six pigs at each time-point. ¹*Differs significantly in ASC or memory B-cell numbers between virulent and attenuated Wa HRV-inoculated groups for the same isotype at the same time-point from the same tissues (Kruskal–Wallis rank sum test, P < 0·05). PBL, peripheral blood lymphocytes.

Figure 3

Isotype-specific antibody-secreting cells (ASC) and memory B cells to Wa human rotavirus (HRV) in gnotobiotic pigs following oral inoculation with live virulent or attenuated Wa HRV and challenge with virulent Wa HRV. Mononuclear cells (MNC) from the duodenum, ileum, mesenteric lymph nodes (MLN), spleen and bone marrow (BM) of pigs were extracted and assayed on postinoculation day (PID) 35 (postchallenge day [PCD] 7). Enzyme-linked immunospot (ELISPOT) assays for determining in vivo HRV-activated antibody-secreting cell (ASC) numbers were performed on the day of MNC extraction. ELISPOT assays for determining memory B-cell numbers were carried out after the MNC had been stimulated in vitro with HRV antigen in cell culture for 5 days. Data represent the mean numbers of Wa HRV-specific ASC or memory B cells per 5 × 10⁵ MNC for three to six pigs at each time-point. *Differs significantly in ASC or memory B-cell numbers between virulent and attenuated Wa HRV-inoculated groups for the same isotype at the same time point from the same tissues; †differs significantly in ASC numbers when compared to PID 28 (PCD 0) for the same isotype from the same tissues in the same group (Kruskal–Wallis rank sum test, P < 0·05). PBL, peripheral blood lymphocytes.

Figure 4

Kinetics of isotype-specific and virus-neutralizing (VN) antibody responses to Wa human rotavirus (HRV) in the serum of pigs inoculated with virulent Wa HRV. Data represent the geometric mean antibody titres (GMT) from two pigs at each time-point.