Therapeutic immune clearance of rabies virus from the CNS

Abstract

The long-held concept that rabies infection is lethal in humans once the causative rabies virus has reached the CNS has been called into question by the recent survival of a number of patients with clinical rabies. Studies in animal models provide insight into why survival from a rabies virus infection that has spread to the CNS is possible and the immune mechanisms involved. In the CNS, both innate mechanisms capable of inhibiting virus replication and the activity of infiltrating rabies virus-specific T and B cells with the capacity to clear the virus are required. Deficiencies in the induction of either aspect of rabies immunity can lead to lethal consequences but may be overcome by novel approaches to active and passive immunization.

Figure 1. Passive administration of rabies virus immune sera from normal mice inhibits CVS-F3 replication in the CNS tissues of B-cell-deficient mice

Groups of five JHD^−/− mice were infected with 10⁵ focus-forming units of CVS-F3 intranasally. A total of 5 days later, mice were injected intraperitoneally with 0.5 ml of either normal or rabies virus immune serum. At the time points indicated, cortex and cerebellar tissues were collected and virus replication was assessed by real-time quantitative RT-PCR. Virus replication is expressed as the mean plus the standard error of the mean copies of rabies virus nucleoprotein mRNA in 50 ng of cDNA isolated from CNS tissues. Statistically significant differences in gene expression between tissues from mice receiving normal and rabies virus immune serum, determined by the Mann–Whitney test, are denoted by *(p < 0.05) and **(p < 0.01).

Figure 2. Antibody-independent control of CVS-F3 replication in the CNS

Groups of normal B6.129, JHD^−/− and Rag-2^−/− mice on a B6.129 background were infected intranasally with 10⁵ focus-forming units of CVS-F3. At the indicated times after infection, rabies virus replication in the CNS was estimated by quantifying the amount of viral nucleoprotein mRNA in CNS tissues using real-time quantitative RT-PCR as detailed previously [26]. Additional groups of ten mice each were followed for survival. RABV: Rabies viruses.

Figure 3. Elevated fluid-phase blood–brain barrier permeability is the dominant outcome of mixed CVS-F3 and silver-haired bat rabies virus infection but occurs too slowly following CVS-F3 infection to have practical therapeutic value

(A) Groups of five to six 129SvEv mice were either left uninfected or were infected with 10⁵ focus-forming units of CVS-F3 intranasally, 10⁴ focus-forming units of SHBRV intradermally in the ear, as previously described [12], or infected using both regimens. The extent of fluid-phase BBB permeability was assessed by measuring leakage of Na-fluorescein from circulation into the cerebellar tissues at 8 days postinfection as detailed previously [18]. Results are expressed as the mean ± S.E.M. (B) Groups of ten 129SvEv mice were infected with 10⁵ focus-forming units of CVS-F3 intranasally and with 10⁴ focus-forming units of SHBRV intradermally in the ear at the indicated intervals. RABV nucleoprotein levels for each virus in brain tissues were determined by real-time quantitative RT-PCR at 8 days after CVS-F3 infection using primers and probes specific for the two viruses. Synthetic nucleoprotein cDNAs were used to quantify the copy numbers of each nucleoprotein mRNA in 100 ng of total brain RNA. **Statistical significance of the differences between uninfected and infected groups was tested using the Mann–Whitney test (p < 0.01). BBB: Blood–brain barrier; RABV: Rabies virus; S.E.M.: Standard error of the mean; SHBRV: Silver-haired bat rabies virus.

Figure 4. An interaction between CD4 T cells and MHC class II-positive cells in the neurovasculature is deficient in mice infected with wild-type rabies virus

(A) Frozen brain sections from mice infected with either CVS-F3 or SHBRV 8 days previously, as described in the legend to Figure 3, were stained with fluorophore-conjugated antibodies specific for MHC class II (red) and CD4 (green), as previously described [17]. (B) RNA isolated from brain microvessels of uninfected or RABV-infected mice was subjected to RT-PCR with primers specific for the gene products indicated, as described elsewhere [24]. The house-keeping gene L-13 was included as a control for the level of mRNA in the samples. SHBRV: Silver-haired bat rabies virus.