Protective vaccine-induced CD4(+) T cell-independent B cell responses against rabies infection

Abstract

A major goal in rabies virus (RV) research is to develop a single-dose postexposure prophylaxis (PEP) that would simplify vaccination protocols, reduce costs associated with rabies prevention in humans, and save lives. Live replication-deficient RV-based vaccines are emerging as promising single-dose vaccines to replace currently licensed inactivated RV-based vaccines. Nonetheless, little is known about how effective B cells develop in response to live RV-based vaccination. Understanding this fundamental property of rabies immunology may help in developing a single-dose RV vaccine. Typically, vaccines induce B cells secreting high-affinity, class-switched antibodies during germinal center (GC) reactions; however, there is a lag time between vaccination and the generation of GC B cells. In this report, we show that RV-specific antibodies are detected in mice immunized with live but not inactivated RV-based vaccines before B cells displaying a GC B cell phenotype (B220(+)GL7(hi)CD95(hi)) are formed, indicating a potential role for T cell-independent and early extrafollicular T cell-dependent antibody responses in the protection against RV infection. Using two mouse models of CD4(+) T cell deficiency, we show that B cells secreting virus-neutralizing antibodies (VNAs) are induced via T cell-independent mechanisms within 4 days postimmunization with a replication-deficient RV-based vaccine. Importantly, mice that are completely devoid of T cells (B6.129P2-Tcrβ(tm1Mom) Tcrδ(tm1Mom)/J) show protection against pathogenic challenge shortly after immunization with a live replication-deficient RV-based vaccine. We show that vaccines that can exploit early pathways of B cell activation and development may hold the key for the development of a single-dose RV vaccine wherein the rapid induction of VNA is critical.

Fig 1

Protection against pathogenic RV challenge soon after immunization with RV-based vaccines. On day 0, mice were immunized with 10⁶ FFU/mouse with rRV-ΔM, rRV, rRV-UV, or PBS and then challenged with 10⁵ FFU/mouse challenge virus strain N2c (CVS-N2c) on day 1, 2, 3, 4, 5, or 6 postimmunization. Data shown are a combination of 2 independent experiments consisting of 5 mice per experiment (n = 10 per group). Kaplan-Meier survival curves were analyzed by the log rank test (*, P < 0.05). The data indicate that live RV-based vaccines elicited protective immune responses within days of immunization, while inactivated RV-based vaccines, which are the vaccines currently used to prevent rabies in humans, do not.

Fig 2

Kinetics of GC B cell development in the lymph nodes and spleens of mice immunized with RV-based vaccines. Groups of 5 female C57BL/6 mice aged 6 to 8 weeks were immunized intramuscularly with a single dose of 10⁶ FFU/mouse of rRV-ΔM, rRV, rRV-UV, or PBS. On the indicated days postimmunization, draining (inguinal) lymph nodes and spleens were removed, single cell suspensions prepared, and cells immunostained to determine the number of GC B cells. (A) Representative gating strategy of live cells gated for total B cells (B220⁺) from mice immunized with rRV (top) or PBS (bottom). (B) Representative gating strategy of total B cells (B220⁺) gated for GL7 and CD95 from mice immunized with rRV (top) or PBS (bottom). (C) Numbers of GC B cells in mice immunized with different RV-based vaccines per 100,000 total live cells in the lymph node. Significant numbers of GC B cells were not detected until 7 days postimmunization with rRV or rRV-ΔM vaccines. Although an increase in the number of GC B cells was detected in mice immunized with rRV-UV by day 7 postimmunization, these numbers were not significantly different from counts observed in mice immunized with PBS alone. (D) Number of GC B cells in mice immunized with the different RV-based vaccines per 100,000 live cells in the spleen. Consistent with the lack of B cell expansion in the spleens of immunized mice (not shown), the number of GC B cells forming in the spleen was minimal, supporting the notion that B cell activation occurs in the lymph node and not the spleen of mice immunized with RV-based vaccines. n ≥ 10/group; to compare two groups of data, we used an unpaired, two-tailed t test; *, P < 0.05.

Fig 3

Early antibody titers induced by RV-based vaccines. Groups of 5 female C57BL/6 mice aged 6 to 8 weeks were immunized intramuscularly with a single dose of 10⁶ FFU/mouse of rRV-ΔM, rRV, rRV-UV, or PBS. On the indicated times postimmunization, RV glycoprotein (G)-specific IgG (A), IgG3 (B), IgG2c (C), IgG1 (D), or IgM (E) antibodies were determined by ELISA. Natural (IgM) and class-switched antibodies were detected as early as 3 to 4 days postimmunization with live RV-based vaccines; however, only IgM was detected in significant amounts in mice immunized with rRV-UV compared to mice immunized with PBS alone at these early time points. n = 5/group; to compare two groups of data, we used an unpaired, two-tailed t test; *, P < 0.05. OD490, optical density at 490 nm.

Fig 4

Early VNA responses in mice immunized with RV-based vaccines. The experiment illustrated in Fig. 3 was repeated, except VNA titers were determined by the rapid fluorescent focus inhibition test (RFFIT) from individual immunized mice. 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-RV antibody reference standard. Virus-neutralizing antibodies were detected in mice immunized with live RV-based vaccines as early as 3 days postimmunization.

Fig 5

Antibody responses in rRV-ΔM-immunized mice depleted of CD4⁺ T cells. C57BL/6 mice were depleted of CD4⁺ T cells using anti-CD4 antibody (GK1.5) as described in Materials and Methods and then immunized intramuscularly with 10⁶ FFU/mouse rRV-ΔM. Sera from immunized mice were collected on the indicated days. Spleens from CD4⁺ T cell-depleted and wild-type mice were collected throughout the sample period and tested for CD4⁺ T cell depletion. (A) Depletion of CD4⁺ T cell-depleted mice (right panel) at all time points, including day 7 postimmunization, was determined to be 95% to 99% compared to nondepleted mice (left panel) as shown by representative analysis of CD4 T cells. (B through E) On the indicated times postimmunization, RV glycoprotein (G)-specific IgG (B), IgM (C), IgG3 (D), and IgG2c (E) antibodies were determined by ELISA. OD490, optical density at 490 nm. (F) Virus-neutralizing antibodies were also determined as described in the legend to Fig. 4. While wild-type mice had higher VNA titers than CD4⁺ T cell-depleted mice, the latter showed antibody titers indicative of a satisfactory immunization (i.e., >0.5 IU/ml) as early as 4 days postimmunization in the absence of CD4⁺ T cell help. IgG (of all subclasses tested) increased over time in CD4⁺ T cell-depleted mice, and IgM antibodies were also detected. n = 5/group of CD4⁺ T cell-depleted mice; n = 4/group of wild-type mice; to compare two groups of data, we used an unpaired, two-tailed t test; *, P < 0.05.

Fig 6

Anti-RV antibody responses and protection against pathogenic challenge in TCRβδ^−/− mice. (A) Groups of 5 female 6- to 8-week-old C57BL/6 or TCRβδ^−/− mice were immunized intramuscularly with 10⁶ FFU/mouse with rRV-ΔM. Groups of 3 female C57BL/6 or TCRβδ^−/− mice immunized with PBS served as controls. Blood was collected on the indicated days and sera analyzed by RFFIT for VNA titers as described for Fig. 4. (B) In two separate, independent experiments, female 6- to 8-week-old C57BL/6 (total of n = 15) or TCRβδ^−/− (total of n = 15) mice were immunized with rRV-ΔΜ. As controls, female 6- to 8-week-old C57BL/6 (total of n = 10) or TCRβδ^−/− (total of n = 5) mice were mock immunized with PBS. Mice were then challenged with 10⁵ FFU/mouse CVS-N2c 5 days later near the peak of VNA titers (detected in TCRβδ^−/− mice as described for panel A). Mice were observed for 4 weeks for clinical signs of rabies and euthanized at the onset of neurological symptoms. ***, P < 0.001; **, P = 0.001 to 0.01; *, P = 0.01 to 0.05.