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.Rotavirus (RV) is the main cause of severe diarrhea in young infants worldwide and is responsible for 611,000 deaths per year (36). Two vaccines based on attenuated live viruses have recently completed successful phase III clinical trials and showed efficacy in preventing severe RV diarrhea caused by multiple serotypes (45, 50). However, the relevant immune effectors required for achieving protection against diverse circulating strains are still a matter of considerable debate. While CD8 ϩ T cells facilitate the timely clearance of rotavirus infection, B cells with the appropriate pattern of mucosal homing receptors and antibody production have been demonstrated as crucial for protection from reinfection (17, 24).Neutralizing antibodies against RV that block viral replication in cell culture are felt to play a major role in defense. A variety of passive antibody transfer studies in animals (28), as well as immunization studies using selected rotavirus reassortants in humans (7), have demonstrated a clear role for VP4 and VP7 in protective immunity. Neutralizing antibodies are directed against the outer shell VP4 and VP7 proteins, whose variable antigenic specificities define multiple serotypes, assigned as P and G serotypes, respectively (41). Worldwide, the G1-to-G4 serotypes associated with P4 and P8 and the more recently described G9/P6 or P8 serotypes are globally important rotavirus G/P combinations in human...

Plasmacytoid dendritic cells promote rotavirus-induced human and murine B cell responses

“…IgA, but not IgG, monoclonal antibodies administered by a hybridoma "backpack" tumor are sufficient to protect naive newborn mice from primary infection (56), while human RV-specific IgA as well as IgG antibodies are able to neutralize RV in cell culture (25). Additionally, RV-specific IgA but not IgG can confer resistance to infection by neutralizing virus intracellularly following transcytosis (40,57,58), and RV-specific serum IgA levels predict protection against severe clinical disease (59).…”

Rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection

“…and triple-layered particles, conferring protection by intracellular neutralization following transcytosis in mice and also in vitro using polarized Caco-2 cells (46)(47)(48). The mechanism by which these antibodies interfere with RV replication is not yet understood, and it is postulated that they may interfere with early replication events by inducing conformational changes in the VP6 layer, or alternatively, the anti-VP6 antibodies may interfere with the secretory pathways during late replication, preventing virus assembly and release (47).…”

“…The mechanism by which these antibodies interfere with RV replication is not yet understood, and it is postulated that they may interfere with early replication events by inducing conformational changes in the VP6 layer, or alternatively, the anti-VP6 antibodies may interfere with the secretory pathways during late replication, preventing virus assembly and release (47). It is also known that not all anti-VP6 antibodies can neutralize RV replication intracellularly (49).…”

Functional Maturation of the Human Antibody Response to Rotavirus

“…Some anti-VP6 IgA mAbs, however, protect nonimmune mice from infection and clear chronic infection in SCID mice (2). The mechanism of the protection mediated by such Abs is still being investigated, but it is thought that they block the virus life cycle at the step of transcription, probably inside virus-infected cells (3). After the virus has entered the cell, the outer capsid is released, exposing the VP6-coated double-layer particle (DLP), which then is activated for transcription.…”

“…After the virus has entered the cell, the outer capsid is released, exposing the VP6-coated double-layer particle (DLP), which then is activated for transcription. These anti-VP6 mAbs block viral transcription in vitro in a dose-dependent manner when introduced into a cell concurrently with DLPs or during transcytosis in the cell (3)(4)(5)(6). The specific role of such VP6 Abs in preventing or resolving human infection is still unclear.…”