Sufficient virus-neutralizing antibody in the central nerve system improves the survival of rabid rats

Abstract

Background: Rabies is known to be lethal in human. Treatment with passive immunity for the rabies is effective only when the patients have not shown the central nerve system (CNS) signs. The blood-brain barrier (BBB) is a complex functional barrier that may compromise the therapeutic development in neurological diseases. The goal of this study is to determine the change of BBB integrity and to assess the therapeutic possibility of enhancing BBB permeability combined with passive immunity in the late stage of rabies virus infection.

Methods: The integrity of BBB permeability in rats was measured by quantitative ELISA for total IgG and albumin levels in the cerebrospinal fluid (CSF) and by exogenously applying Evans blue as a tracer. Western blotting of occludin and ZO-1, two tight junction proteins, was used to assess the molecular change of BBB structure.The breakdown of BBB with hypertonic arabinose, recombinant tumor necrosis factor-alpha (rTNF-γ), and focused ultrasound (FUS) were used to compare the extent of BBB disruption with rabies virus infection. Specific humoral immunity was analyzed by immunofluorescent assay and rapid fluorescent focus inhibition test. Virus-neutralizing monoclonal antibody (mAb) 8-10E was administered to rats with hypertonic breakdown of BBB as a passive immunotherapy to prevent the death from rabies.

Results: The BBB permeability was altered on day 7 post-infection. Increased BBB permeability induced by rabies virus infection was observed primarily in the cerebellum and spinal cord. Occludin was significantly decreased in both the cerebral cortex and cerebellum. The rabies virus-specific antibody was not strongly elicited even in the presence of clinical signs. Disruption of BBB had no direct association with the lethal outcome of rabies. Passive immunotherapy with virus-neutralizing mAb 8-10E with the hypertonic breakdown of BBB prolonged the survival of rabies virus-infected rats.

Conclusions: We demonstrated that the BBB permeability was altered in a rat model with rabies virus inoculation. Delivery of neutralizing mAb to the infected site in brain combined with effective breakdown of BBB could be an aggressive but feasible therapeutic mode in rabies when the CNS infection has been established.

Figure 1

BBB permeability was altered in the CSF of rats infected with rabies virus. BBB permeability was assessed by measuring (A) albumin and (B) total IgG from the CSF after rabies virus infection and expressed as the concentration (μg/ml) (n = 8). Statistical significance of differences in permeability was calculated using Student’s t-test and are denoted by the symbol *p < 0.05. The CSF to serum ratios were presented in (C) for albumin and (D) for IgG. Each dot represented one individual rat.

Figure 2

Localization of BBB permeability changes in the brain of rabies virus-infected rats. Brains were removed from Evans blue-treated rats either in the control or rabies virus-infected animals on day 10 to 11 p.i.. BBB permeability was assessed by measuring the EB content which was determined by a standard curve. Compared with controls (n = 4), rabies virus-infected rats (n = 8) showed a significant increased in the amount of EB (*p < 0.05).

Figure 3

Expression of tight junction protein, occludin, was decreased in the brains of rabies-infected rats. Western blotting analyses of tight junction proteins, occludin and ZO-1, were shown in (A) the cerebral cortex and cerebellum. Relative levels of tight junction protein (B) occludin and (C) ZO-1 expression were determined by densitometric analysis. All samples analyzed (n = 4) were normalized to the intensity of corresponding β-actin bands. Results are expressed as mean ± SEM. *p < 0.05, significantly different from the control rats.

Figure 4

Alteration of BBB permeability was compared in three methods. BBB permeability was assessed by measuring the albumin (A, C, E) and total IgG (B, D, F) contents from the CSF after hypertonic arabinose (n = 8), rTNF-α (n = 5) and FUS (n = 5) treatment. The data were expressed as the concentration (μg/ml). Statistical significance of differences in BBB permeability was calculated using Student’s t-test and are denoted by the symbol *p < 0.05.

Figure 5

Survival percentage of rabies virus-infected rats infused with or without BBBO. Groups of rats (n = 7) after rabies virus (RV) infection were infused with or without hyperosmotic arabinose to disrupt BBB on day 7 and 9 p.i. and assessed the morbidity and mortality. BBBO was performed as described in the Methods. Comparison between RV alone and RV plus BBBO showed no significant difference between each groups (p > 0.05).

Figure 6

Rabies virus-specific antibody productions were slightly elicited after infection. Kinetics of (A) specific IgG and (B) virus-neutralizing antibody (VNA) production in the sera and CSF of rabies virus-infected rats (n = 8) were assessed by IFA and RFFIT, respectively. Levels of antibodies are presented as the geometric mean ± SEM of the last dilution of serum and CSF.

Figure 7

Survival of rabies virus-infected rats treated with passive immunotherapy alone or in combination with BBBO. (A) Diagram of treatment schedule: on day 7 and 9 p.i., group 1 animals received mAb 8-10E alone (n = 7) and group 2 animals received mAb 8-10E + BBBO (n = 8), (B) Survival of rats following infection with four different treatment: (1) 8-10E alone on day 7 p.i., (2) 8-10E + BBBO on day 7 p.i., (3) 8-10E alone on day 9 p.i., and (4) 8-10E + BBBO on day 9 p.i.. Rats were monitored for morbidity and mortality for 40 days. 8-10E: indicated that rats was injected with virus-neutralizing mAb 8-10E. BBBO: indicated that the BBB permeability was enhanced with hypertonic arabinose infusion. Statistical significance of differences in survival rats was denoted by the symbol *p<0.05. (C) Detection of viral RNA in the brain tissues infected with rabies virus. Viral nucleoprotein mRNA expression was detected in the cerebrum, cerebellum and spinal cord of rats died of BBBO and mAb 8-10E + BBBO treatment on day 14 p.i. (numbered 1, 2, 3, 4). The size of the amplified fragment was estimated to be 777 bp. No viral genome was detected in the brain tissues and spinal cord of rats survived for 40 day p.i. (numbered 5, 6, 7, 8). The lane marked “P” is the positive control taken from a cultured virus suspension.