Germ Line IgM Is Sufficient, but Not Required, for Antibody-Mediated Alphavirus Clearance from the Central Nervous System

Abstract

Sindbis virus (SINV) infection of neurons in the brain and spinal cord in mice provides a model system for investigating recovery from encephalomyelitis and antibody-mediated clearance of virus from the central nervous system (CNS). To determine the roles of IgM and IgG in recovery, we compared the responses of immunoglobulin-deficient activation-induced adenosine deaminase-deficient (AID^-/-), secretory IgM-deficient (sIgM^-/-), and AID^-/- sIgM^-/- double-knockout (DKO) mice with those of wild-type (WT) C57BL/6 mice for disease, clearance of infectious virus and viral RNA from brain and spinal cord, antibody responses, and B cell infiltration into the CNS. Because AID is essential for immunoglobulin class switch recombination and somatic hypermutation, AID^-/- mice produce only germ line IgM, while sIgM^-/- mice secrete IgG but no IgM and DKO mice produce no secreted immunoglobulin. After intracerebral infection with the TE strain of SINV, most mice recovered. Development of neurologic disease occurred slightly later in sIgM^-/- mice, but disease severity, weight loss, and survival were similar between the groups. AID^-/- mice produced high levels of SINV-specific IgM, while sIgM^-/- mice produced no IgM and high levels of IgG2a compared to WT mice. All mice cleared infectious virus from the spinal cord, but DKO mice failed to clear infectious virus from brain and had higher levels of viral RNA in the CNS late after infection. The numbers of infected cells and the amount of cell death in brain were comparable. We conclude that antibody is required and that either germ line IgM or IgG is sufficient for clearance of virus from the CNS.IMPORTANCE Mosquito-borne alphaviruses that infect neurons can cause fatal encephalomyelitis. Recovery requires a mechanism for the immune system to clear virus from infected neurons without harming the infected cells. Antiviral antibody has previously been shown to be a noncytolytic means for alphavirus clearance. Antibody-secreting cells enter the nervous system after infection and produce antiviral IgM before IgG. Clinical studies of human viral encephalomyelitis suggest that prompt production of IgM is associated with recovery, but it was not known whether IgM is effective for clearance. Our studies used mice deficient in production of IgM, IgG, or both to characterize the antibody necessary for alphavirus clearance. All mice developed similar signs of neurologic disease and recovered from infection. Antibody was necessary for virus clearance from the brain, and either early germ line IgM or IgG was sufficient. These studies support the clinical observation that prompt production of antiviral antibody is a determinant of outcome.

Keywords: Sindbis virus; antibody; mice; neurons; viral encephalomyelitis; virus clearance.

FIG 1

Morbidity and mortality of SINV-infected C57BL/6 (B6) mice. WT (n = 22), AID^−/− (n = 31), sIgM^−/− (n = 33), and DKO (n = 29) mice were inoculated intracerebrally with 1,000 PFU SINV TE and followed daily for the onset of clinical signs (A) (**, P < 0.01, Kaplan-Meier log-rank test), clinical scores (B) (*, P < 0.05; ***, P < 0.001 for AID^−/− versus sIgM^−/− mice, 2-way analysis of variance with Tukey's multiple-comparison test), percent body weight change (C), and survival (D).

FIG 2

Virus clearance from brain and spinal cord. WT, AID^−/−, sIgM^−/−, and DKO mice were inoculated intracerebrally with 1,000 PFU SINV TE, and brain (A) and spinal cord (B) homogenates from 3 mice from each group at each time point were assayed for infectious virus by plaque assay. RNA was extracted from brain (C) and spinal cord (D) tissues from 6 to 8 mice from each group at each time point and assayed for viral RNA by RT-qPCR. P values were determined by 2-way analysis of variance with Bonferroni's posttest. *, P < 0.05; **, P < 0.01, ***, P < 0.001.

FIG 3

Distribution of viral protein, RNA, and cell death in brain. WT, AID^−/−, sIgM^−/−, and AID^−/− sIgM^−/− (DKO) mice were inoculated intracerebrally with 1,000 PFU SINV TE. Formalin-fixed paraffin-embedded brain slices were stained by use of a combination of RNA fluorescence in situ hybridization (FISH) for SINV E1 and E2 genes (red) and fluorescent antibody for SINV proteins (white) and by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL-TSA; green) for dead cells in the presence of DAPI. (A) Brain section from a WT mouse 5 days after infection showing individual channels and merged channels; (B) merged images of brains from WT, AID^−/−, sIgM^−/−, and DKO mice collected 5, 7, and 10 days (D) after infection.

FIG 4

Quantification of cells positive for viral protein, RNA, and TUNEL staining in brain. WT, AID^−/−, sIgM^−/−, and AID^−/− sIgM^−/− (DKO) mice were inoculated intracerebrally with 1,000 PFU SINV TE. Formalin-fixed paraffin-embedded brain slices from 5, 7, and 10 days after infection were stained by use of a combination of RNA fluorescence in situ hybridization (FISH) for SINV E1 and E2 genes and antibody for SINV proteins (IF) and by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL-TSA) for dead cells (Fig. 3). The numbers of cells positive for SINV protein (A), viral RNA (B), and TUNEL-TSA (C) per slice of right brain hemisphere are shown. Data are from one of two experiments and are for 3 mice per group per time point. *, P < 0.05; **, P < 0.01.

FIG 5

Ifng and Aid mRNA expression after infection. WT, AID^−/−, sIgM^−/−, and AID^−/− sIgM^−/− (DKO) mice were inoculated intracerebrally with 1,000 PFU SINV TE. RNA was extracted from brain and spinal cord homogenates, and RT-qPCR was performed to determine the change (fold increase) in Ifng mRNA expression in brains (A) and spinal cords (B) from that before infection (day 0). Because AID^−/− and DKO mice do not express the AID gene, only WT and sIgM^−/− mice were tested for Aid mRNA expression in brains (C) and spinal cords (D). *, P < 0.05; ***, P < 0.001.

FIG 6

Production of SINV-specific IgM and IgG. WT, AID^−/−, sIgM^−/−, and AID^−/− sIgM^−/− (DKO) mice were inoculated intracerebrally with 1,000 PFU SINV TE. Serum and brain and spinal cord homogenates were tested by EIA for SINV-specific IgM (A to C) and IgG (D to F). The graphs show the optical density (OD) of mouse serum (1:100 dilution) (A and D), 20% brain homogenates (1:8 dilution) (B and E), and 10% spinal cord homogenates (1:4 dilution) (C, F). P values represent the significance of differences between WT and AID^−/− mice for IgM (A to C) and WT and sIgM^−/− mice for IgG (D to F) and were determined by 2-way analysis of variance. Asterisks show significant differences at particular time points (**, P < 0.01, ***, P < 0.001).

FIG 7

Production of SINV-specific IgG isotypes. WT and sIgM^−/− mice were inoculated intracerebrally with 1,000 PFU SINV TE. The levels of each IgG subclass in serum (1:100 dilution), 20% brain homogenates (1:8 dilution), and 10% spinal cord homogenates (1:4 dilution) reactive with SINV by EIA are shown. (A) IgG1; (B) IgG2a; (C) IgG2b; (D) IgG3. P values represent the significance of the differences between mouse groups and were determined by 2-way analysis of variance. Asterisks show significant differences at particular time points (*, P < 0.05; **, P < 0.01, ***, P < 0.001).

FIG 8

Localization and quantification of IgM- and IgG-secreting cells in brain. (A) A 4-by-4 tile scan image (stitching of a 7-by-7 tile scan with 50% overlap) of the right hemisphere of a WT mouse at 10 days after infection shows the distribution of cells with viral proteins (white), IgG (red), and IgM (green). (Inset) A higher-power image shows IgG- and IgM-positive cells infiltrating the pia mater and brain parenchyma near infected cells. (B, C) Numbers of IgM-positive cells (B) and IgG-positive cells (C) per slice of right brain hemisphere (3 mice per group per time point) at 10 days after infection.