Rotavirus anti-VP6 secretory immunoglobulin A contributes to protection via intracellular neutralization but not via immune exclusion

Abstract

Immunoglobulin A (IgA) monoclonal antibodies (MAbs) directed at the conserved inner core protein VP6 of rotavirus, such as the IgA7D9 MAb, provide protective immunity in adult and suckling mice when delivered systemically. While these antibodies do not have traditional in vitro neutralizing activity, they could mediate their antiviral activity either by interfering with the viral replication cycle along the IgA secretory pathway or by acting at mucosal surfaces as secretory IgA and excluding virus from target enterocytes. We sought to determine the critical step at which antirotaviral activity was initiated by the IgA7D9 MAb. The IgA7D9 MAb appeared to directly interact with purified triple-layer viral particles, as shown by immunoprecipitation and immunoblotting. However, protection was not conferred by passively feeding mice with the secretory IgA7D9 MAb. This indicates that the secretory IgA7D9 MAb does not confer protection by supplying immune exclusion activity in vivo. We next evaluated the capacity of polymeric IgA7D9 MAb to neutralize rotavirus intracellularly during transcytosis. We found that when polymeric IgA7D9 MAb was applied to the basolateral pole of polarized Caco-2 intestinal cells, it significantly reduced viral replication and prevented the loss of barrier function induced by apical exposure of the cell monolayer to rotavirus, supporting the conclusion that the antibody carries out its antiviral activity intracellularly. These findings identify a mechanism whereby the well-conserved immunodominant VP6 protein can function as a target for heterotypic antibodies and protective immunity.

FIG. 1.

Western blot analysis of the pIgA7D9 MAb, SIgA7D9 MAb, and SC preparations. Polymeric IgA7D9 MAb, reconstituted SIgA7D9 MAb, and SC were purified by size-exclusion chromatography and analyzed by Western blotting under nonreducing conditions. (A) pIgA7D9 and SIgA7D9 MAbs were revealed with horseradish peroxidase-coupled anti-murine IgA serum. (B) Both polymeric forms contained the J chain, reflecting proper assembly. (C) Expected covalent and noncovalent association of SC and pIgA7D9 MAb was confirmed upon immunodetection with anti-SC antiserum. Molecular mass markers (kDa) are indicated alongside the sample lanes.

FIG. 2.

Interaction between RV TLP and pIgA7D9 or SIgA7D9 MAbs. Immunoprecipitation (IP) of RV TLP was performed with two quantities (50 μg and 500 μg) of pIgA7D9 MAb and equimolar amounts (62.5 μg and 625 μg) of SIgA7D9 MAb. Following protein separation by SDS-PAGE, detection of VP8* by specific 5.73 MAb indicates that TLP are recognized by the two molecular forms of the anti-VP6 MAb, namely pIgA7D9 MAb (lanes 1 and 2) and SIgA7D9 MAb (lanes 3 and 4). No signal was observed in the absence of IgA7D9 MAb (lane 5) or with 500 μg of irrelevant pIgAC5 MAb (lane 6).

FIG. 3.

Oral feeding with SIgA7D9 does not reduce viral shedding. (A) Time frame summarizing the kinetics of i.p. and oral passive administration of the various molecular forms of pIgA7D9 and SIgA7D9. (B) Mean percentages of reduction of virus shedding (± standard error) compared to the PBS group are reported from BALB/c mice inoculated i.p. with pIgA7D9 MAb or dosed orally with SIgA7D9, pIgA7D9 MAb, and SC. A statistically significant difference was only achieved for viral shedding in the group receiving pIgA7D9 MAb i.p. (Student's t test, P < 0.005). Similar results were obtained in two other independent experiments. (C) Level of IgA7D9 MAb in mouse feces measured 2 h after passive administration of pIgA7D9 MAb i.p. or SIgA7D9 MAb orally. BD, below the level of detection.

FIG. 4.

Intracellular neutralization of RRV by pIgA7D9 MAb. (A) Stability of the Caco-2 monolayer TER exposed to different virus MOI as a function of time. (B) Transcytosis of the various IgAs used in this study and molecular forms thereof across Caco-2 cell monolayers was measured by ELISA as the appearance of SIgA in the apical chamber 4 and 20 h after addition of the purified IgA MAbs in the basolateral compartment (B). (C) The amount of IgA present in the basolateral chamber (5 μg/ml) is not limiting in the assay, as measured at 20 h.