Intracellular neutralisation of rotavirus by VP6-specific IgG

Abstract

Rotavirus is a major cause of gastroenteritis in children, with infection typically inducing high levels of protective antibodies. Antibodies targeting the middle capsid protein VP6 are particularly abundant, and as VP6 is only exposed inside cells, neutralisation must be post-entry. However, while a system of poly immune globulin receptor (pIgR) transcytosis has been proposed for anti-VP6 IgAs, the mechanism by which VP6-specific IgG mediates protection remains less clear. We have developed an intracellular neutralisation assay to examine how antibodies neutralise rotavirus inside cells, enabling comparison between IgG and IgA isotypes. Unexpectedly we found that neutralisation by VP6-specific IgG was much more efficient than by VP6-specific IgA. This observation was highly dependent on the activity of the cytosolic antibody receptor TRIM21 and was confirmed using an in vivo model of murine rotavirus infection. Furthermore, mice deficient in only IgG and not other antibody isotypes had a serious deficit in intracellular antibody-mediated protection. The finding that VP6-specific IgG protect mice against rotavirus infection has important implications for rotavirus vaccination. Current assays determine protection in humans predominantly by measuring rotavirus-specific IgA titres. Measurements of VP6-specific IgG may add to existing mechanistic correlates of protection.

Fig 1. Intracellular neutralisation assay.

(A) Images of rotavirus-infected cells following electroporation of anti-VP6 MAb. Rotavirus-infected cells were stained with sheep polyclonal anti-rotavirus antibody and subsequently Alexa-Fluor 488-conjugated anti-sheep Ig. Scale bar 200μm (B) Fluorescent foci in (A) captured on a Nikon Eclipse Ti microscope were quantified by NIS analysis software. (C) Confocal images of a rotavirus-infected murine embryonic fibroblast cell 1 hour post infection in the presence of electroporated anti-VP6 Mab, scale bar 10μm. (D) Comparison of extracellular versus intracellular neutralisation for two anti-VP6 MAbs, 7D9 and 2C5. (E) Testing for intracellular neutralisation mediated by MAbs recognising VP4 (7A12 and 1A9).

Fig 2. Analysis of VP6-specific polyclonal immune responses in mice and humans

(A) Intracellular neutralisation of rotavirus infection by anti-DLP polyclonal serum versus control serum electroporated into cells. (B) Faecal antigen shedding as detected by ELISA on days 1–7 post infection in mice immunized with DLPs 2 and 4 weeks prior to infection, compared to naive mice (4–5 mice per group). (C) ELISAs to detect IgG and IgA polyclonal antibodies raised by DLP immunization in mice, each symbol representing one mouse. (D) Intracellular neutralisation of rotavirus (strain SA11) by VP6-specific IgG purified from human pooled IgG in MA104 cells. For all graphs, error bars represent standard error, **** p = < 0.0001, ** p = < 0.01, * p = < 0.05, NS not significant.

Fig 3. Production and characterization of VP6-specific recombinant monoclonal antibodies.

(A) Mouse-human chimeric 7D9 hIgG binds to DLPs by ELISA while control antibody (9C12) does not. (B) Mouse-human chimeric 7D9 hIgA binds to DLPs by ELISA. (C) Intracellular neutralisation of rotavirus by VP6-specific 7D9 with human IgG1 Fc region compared to IgA1 region electroporated into cells. For all graphs, error bars represent standard error, **** p = < 0.0001, *** p = < 0.001, ** p = < 0.01, * p = < 0.05, NS not significant.

Fig 4. Mechanisms of antibody-mediated intracellular neutralisation.

(A) Intracellular neutralisation of rotavirus by VP6-specific IgA in wild type (WT) and TRIM21 knockout (TRIM21KO) MA104 cells. (B) Intracellular neutralisation by VP6-specific IgG in WT and TRIM21KO knockout MA104 cells. (C) Intracellular neutralisation by WT or non-binding TRIM21 IgG (H433A). (D) Amounts of EDIM virus shedding three days post infection as detected by faecal antigen ELISA in naïve and pre-DLP-immunized, WT (9–10 mice per group) as well as TRIM21KO mice (5 mice per group). (E) EDIM shedding on days 1–7 post infection in WT and TRIM21KO BALB/c mice pre-immunized with DLPs. Horizontal lines represent mean and standard error, *** p = < 0.001, * p = < 0.05, NS not significant.

Fig 5. Rotavirus infection in FcRn deficient mice.

(A) DLP-specific antibodies in serum of naïve and DLP-immunized, WT and FcRnKO mice (5 mice per group, each symbol representing one mouse). (B) Faecal antigen shedding on days 0–6 post infection as detected by ELISA in the mice pre-immunized with DLPs, comparing WT with FcRn knockout (FcRnKO) mice. (C) Amounts of EMcN virus shedding in faeces of the naïve and DLP-immunized, WT and FcRnKO mice, days 4 and 5 of experiment shown in panel (B). Horizontal lines represent mean and standard error, **** p = < 0.0001, * p = < 0.05, NS not significant.