The observation that individuals dying of rabies often have virus-neutralizing antibodies in their circulation suggests that the mere presence of antiviral immunity may not be enough to protect an individual from the disease (14
). This is consistent with the fact that rabies postexposure prophylaxis, which is a combination of active and passive immunization, fails to protect individuals from a lethal outcome when administered after clinical signs of rabies appear (6
). We have previously provided evidence that the failure to clear SHBRV from the CNS tissues of 129/SvEv mice may be a consequence of the maintenance of BBB integrity, which prevents circulating RV-specific immune effectors from reaching infected CNS tissues (23
). In the current study, we confirm this hypothesis by showing that increasing BBB permeability facilitates the infiltration of immune effectors into the infected CNS, thereby reducing the lethality of SHBRV infection.
The development of enhanced BBB permeability and a CNS inflammatory response is required for the clearance of attenuated RV from the infected CNS (21
). Mice with an SJL background are known to develop more extensive CNS inflammation than mice of other backgrounds when subjected to similar stimuli (22
). Notably, with respect to the current investigation, mice with an SJL background more often survive infection with pathogenic RV variants than other mouse strains (16
). We found that a high percentage of PLSJL mice indeed survive infection with a dose of SHRBV that is uniformly lethal for 129/SvEv mice. More importantly, the ability of PLSJL mice to survive the infection is associated with their greater capacity to mediate BBB permeability changes and deliver immune effectors to the CNS. Enhancement in BBB permeability and invasion of immune effectors in 129/SvEv mice infected with attenuated RV occurs primarily in the cerebellum (21
). Consistent with this observation, in the current study, PLSJL mice only developed significantly enhanced BBB permeability in the cerebellum following SHBRV infection (Fig. ).
Despite their natural resistance to lethal SHBRV infection, a significant proportion of SHBRV-infected PLSJL mice die of rabies. At day 8 p.i., mice with clinical signs of rabies that are destined to die invariably had higher virus titers in CNS tissues than their similarly infected but healthy counterparts that often survive. Differences in the extent of the CNS adaptive immune response are unlikely to be responsible for the variation in the levels of virus in the CNS of dying and healthy mice for several reasons: (i) the accumulation of mRNAs specific for immune cell subsets does not significantly differ between these two groups, (ii) similar variability is seen in the absence of immune cell infiltration in SHBRV-infected 129/SvEv mice at day 8 p.i., and (iii) variable levels of virus replication are seen in the CNS tissues of PLSJL mice at day 5 p.i., which is prior to the onset of BBB permeability changes (A. Roy and D. C. Hooper, unpublished data). Thus, for reasons that are not fully understood, the extent of virus replication in the CNS tissues of the mice varies with high levels predictive of death. Consequently, we speculate that at day 8 p.i., the levels of virus in the CNS tissues of some of the SHBRV-infected PLSJL mice are too high for the infiltrating immune effectors to clear.
If a proportion of SHBRV-infected PLSJL mice die because virus replication overwhelms the immune effectors reaching the CNS, enhancing immune effector infiltration into the CNS tissue may be expected to increase the capacity of the animal to clear the virus and improve survival. To test this hypothesis, we used an autoimmune CNS inflammatory response to increase BBB permeability and immune cell infiltration into the CNS tissues of SHBRV-infected PLSJL mice. This resulted in a higher percentage of the mice surviving despite the likelihood that some died of autoimmune encephalomyelitis. This confirms that the clearance of SHBRV from the CNS is primarily limited by the delivery of immune effectors to the CNS tissues rather than that the extent of the antiviral response that develops in the periphery and promoting immune effector infiltration into the CNS tissues of the SHBRV-infected host is therapeutic.
Virus replication in the CNS can stimulate the production of steroid hormones through effects on the HPA axis (4
). It is known that pathogenic strains of RV infect the hypothalamus and alter HPA activity (24
), resulting in the elevated production of steroid hormones and the reduced production of growth hormones (29
). These changes may be responsible for the lymphoid organ atrophy and altered immune function (29
) seen during the late stages of rabies. Compared to other strains, mice with an SJL background (i) have reduced HPA axis activity (2
), (ii) develop more extensive CNS inflammation in response to various stimuli (2
), and (iii) are resistant to lethal infection with RV (16
). These facts led us to speculate that the survival of PLSJL mice from a normally lethal dose of SHBRV may be due, at least in part, to the reduced production of steroid hormones as a result of a blunted HPA axis activity. DHEA is particularly interesting in this regard, as it is one of the most abundant hormones produced under HPA axis control (19
) and is known to inhibit CNS inflammation (10
). Administration of DHEA to PLSJL mice, while having no effect on the ability to produce RV-specific IgG, limited BBB permeability elevation and caused increased mortality following SHBRV infection. These findings suggest that the higher levels of DHEA expected in mice with normal HPA axis activity may contribute to increased SHBRV lethality by reducing BBB permeability changes.
Our findings indicate that the delivery of immune effectors to the CNS can reverse the outcome of an otherwise lethal RV infection even after the virus has reached the CNS. Certainly the induction of autoimmune CNS inflammation to open the BBB and deliver RV-specific immune effectors to CNS tissues is not an appropriate therapy for humans. However, the utilization of a mechanism to enhance BBB permeability that is unaffected by SHBRV infection may be crucial to the rescue of an individual who has been diagnosed with rabies based on the appearance of clinical signs. We expect that this may also apply to other CNS diseases where the delivery of therapeutic agents across the BBB is necessary.