Reinvestigating the role of IgM in rabies virus postexposure vaccination

Abstract

B cells secreting IgG antibodies, but not IgM, are thought to be solely responsible for vaccine-induced protection against rabies virus (RABV) infections in postexposure settings. In this report, we reinvestigated the potential for IgM to mediate protection in a mouse model of RABV vaccination. Immunocompetent mice immunized with an experimental live replication-deficient RABV-based vaccine produced virus neutralizing antibodies (VNAs) within 3 days of vaccination. However, mice unable to produce soluble IgM (sIgM(-/-)) did not produce VNAs until 7 days postimmunization. Furthermore, sIgM(-/-) mice were not protected against RABV infection when challenged 3 days postimmunization, while all wild-type mice survived challenge. Consistent with the lack of protection against pathogenic RABV challenge, approximately 50- to 100-fold higher viral loads of challenge virus were detected in the muscle, spinal cord, and brain of immunized sIgM(-/-) mice compared to control mice. In addition, IgG antibody titers in vaccinated wild-type and sIgM(-/-) mice were similar at all time points postimmunization, suggesting that protection against RABV challenge is due to the direct effects of IgM and not the influence of IgM on the development of effective IgG antibody titers. In all, early vaccine-induced IgM can limit dissemination of pathogenic RABV to the central nervous system and mediate protection against pathogenic RABV challenge. Considering the importance for the rapid induction of VNAs to protect against RABV infections in postexposure prophylaxis settings, these findings may help guide the development of a single-dose human rabies vaccine.

Fig 1

Vaccine-induced IgM helps to protect mice against pathogenic RABV challenge. sIgM^−/− or wild-type control mice were immunized i.m. with 10⁶ FFU of rRABV-ΔM or PBS alone and then challenged 3 days later i.m. with 10⁵ FFU of pathogenic RABV CVS-N2c. Survivorship (A) and weights (B) were measured and recorded for 27 days postchallenge. Data shown are a combination of 2 independent experiments consisting of 5 mice per experiment (total n = 10 mice/group). Kaplan-Meier survival curves were analyzed by the log-rank test (***, P < 0.001; **, P = 0.001 to 0.01; *, P = 0.01 to 0.05). Symbols for control (PBS-immunized) sIgM^−/− mice were shifted slightly in panel A for clarity. Crosses in panel B indicate the days when the last mice in a specific group were to be euthanized.

Fig 2

Vaccine-induced IgM helps to prevent the spread of pathogenic RABV into the CNS. Groups of sIgM^−/− or wild-type mice were immunized with rRABV-ΔM and then challenged with CVS-N2c as described in the legend to Fig. 1. Five days postchallenge, total RNA was isolated from the muscle (A), the spinal cord (B), and the brain (C) and analyzed by qRT-PCR for the presence of CVS-N2c-specific viral mRNA. Data represent a combination of two independent experiments (total n = 8 mice/group). One-way analysis of variance (ANOVA) was used to evaluate statistical significance. Where significant differences were observed, Tukey's multiple comparison test was used to identify significant differences between individual groups (*, P < 0.05).

Fig 3

Production of rRABV-ΔM-induced RABV-specific VNAs is delayed in sIgM^−/− mice compared to wild-type controls. Groups of sIgM^−/− or wild-type mice were immunized i.m. with a single dose of 10⁶ FFU of rRABV-ΔM. Blood was collected on the indicated days postimmunization, and VNA titers from pooled sera were determined using the RFFIT. Neutralization titers, defined as the inverse of the highest serum dilution that neutralizes 50% of the challenge virus (challenge virus strain 11), were normalized to international units/ml (IU/ml) using the WHO anti-RABV antibody reference standard. Data represent 2 independent experiments consisting of sera from 3 mice/experiment pooled and then analyzed in duplicate (total n = 6 mice/group). To compare two groups of data for antibody responses, we used an unpaired, two-tailed t test.

Fig 4

Kinetic analyses of rRABV-ΔM-induced IgM and IgG antibody titers in sIgM^−/− mice. Sera collected from individual mice described in the legend to Fig. 3 were analyzed by ELISA to determine anti-RABV G IgM (A) or IgG (B) antibody titers on the indicated days postimmunization. Data represent two independent experiments (total n = 6 mice/group). For multigroup comparisons, ANOVA was used. Where significant differences were observed, Tukey's multiple comparison test was used to identify significant differences between individual groups. Three (P < 0.001), two (P = 0.001 to 0.01), and one (P = 0.01 to 0.05) asterisk indicates a significant difference between two data points (ns, not significant). OD490, optical density at 490 nm.