Protective immunity to rotavirus (RV) is primarily mediated by antibodies produced by RV-specific memory B cells (RV-mBc).Of note, most of these cells express IgM, but the function of this subset is poorly understood. Here, using limiting dilution assays of highly sort-purified human IgM ؉ mBc, we found that 62% and 21% of total (non-antigen-specific) IgM ؉ and RV-IgM ؉ mBc, respectively, switched in vitro to IgG production after polyclonal stimulation. Moreover, in these assays, the median cloning efficiencies of total IgM ؉ (17%) and RV-IgM ؉ (7%) mBc were lower than those of the corresponding switched (IgG ؉ IgA ؉ ) total (34%) and RV-mBc (17%), leading to an underestimate of their actual frequency. In order to evaluate the in vivo role of IgM ؉ RV-mBc in antiviral immunity, NOD/Shi-scid interleukin-2 receptor-deficient (IL-2R␥null ) immunodeficient mice were adoptively transferred highly purified human IgM ؉ mBc and infected with virulent murine rotavirus. These mice developed high titers of serum human RV-IgM and IgG and had significantly lower levels than control mice of both antigenemia and viremia. Finally, we determined that human RV-IgM ؉ mBc are phenotypically diverse and significantly enriched in the IgM hi IgD low subset. Thus, RV-IgM ؉ mBc are heterogeneous, occur more frequently than estimated by traditional limiting dilution analysis, have the capacity to switch Ig class in vitro as well as in vivo, and can mediate systemic antiviral immunity.

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Circulating human IgM expressing memory B cells have been incompletely characterized. Here, we compared the phenotype and in vitro functional response (capacity to proliferate and differentiate to antibody secreting cells) in response to CpG and a cytokine cocktail (IL-2, IL-6, and IL-10) of sorted naïve B cells, IgM memory B cells and isotype-switched circulating memory B cells. Compared to naïve B cells, IgM memory B cells had lower integrated mean fluorescence intensity (iMFI) of BAFF-R, CD38, CD73, and IL-21R, but higher iMFI of CD95, CD11c, TLR9, PD-1, and CD122. Compared to switched memory B cells, IgM memory B cells had higher iMFI of BAFF-R, PD-1, IL-21R, TLR9, and CD122, but lower iMFI of CD38, CD95, and CD73. Four days after receiving the CpG/cytokine cocktail, higher frequencies of IgM than switched memory B cells—and these in turn greater than naïve cells—proliferated and differentiated to antibody secreting cells. At this time point, a small percentage (median of 7.6%) of stimulated IgM memory B cells changed isotype to IgG. Thus, among the heterogeneous population of human circulating IgM memory B cells a subset is capable of a rapid functional response to a CpG/cytokine stimulus in vitro.

Rotavirus (RV)-specific secretory immunoglobulin (RV-SIg) has been previously detected in serum of naturally RV infected children and shown to reflect the intestinal Ig immune response. Total plasma SIgA and plasma RV-SIg were evaluated by ELISA in children with gastroenteritis due or not due to RV infection and in 50 children vaccinated with the attenuated RIX4414 human RV vaccine and 62 placebo recipients. RV-SIg was only detected in children with evidence of previous RV infection or with acute RV gastroenteritis. Vaccinees had higher RV-SIg titers than placebo recipients and RV-SIg titers increased after the second vaccine dose. RV-SIg measured after the second dose correlated with protection when vaccinees and placebo recipients were analyzed jointly. RV-SIg may serve as a valuable correlate of protection for RV vaccines.