Neurotropic viruses that are antigenically related but differ in pathogenicity, as well as the ability to induce a protective immune response during an infection, provide unique tools to probe the relationship between viral neuropathogenicity and immunogenicity. RVs, which exist as a range of antigenically related variants that are highly diverse in pathogenicity, are particularly useful for such studies. CVS-F3, an RV variant attenuated by a single amino acid substitution in the glycoprotein of CVS (11
), is cleared from the CNS of infected, immunocompetent mice as BBB permeability becomes enhanced and immune effectors accumulate in the infected tissues (21
). Although CVS-F3 spreads extensively through the cortex and cerebellum, BBB permeability changes, as well as CD4 and B-cell infiltration, are considerably higher in the cerebellum, suggesting that the delivery of antiviral effectors is predominantly through the BBB of the cerebellum (21
). This is supported by the present findings that mice infected with SHBRV-17, a pathogenic RV obtained from a human victim of rabies, while developing strong virus-specific immunity in the periphery, fail to develop increased BBB permeability in the cerebellum with its associated CNS inflammatory response and succumb to rabies. Notably, CNS inflammation is seldom seen in animals infected with pathogenic strains of RV, including other SHBRV variants (19
), as well as in postmortem studies of human rabies victims (20
). Since the development of RV-specific immunity in the periphery of CVS-F3- and SHBRV-17-infected mice is comparable, the reduced CNS inflammatory response to RV seen in the latter evidently does not result from a major deficit in immunity. It is possible that IFN-γ-positive cells accumulate to higher levels in the circulation of SHBRV-17-infected animals because of a diminished ability to infiltrate CNS tissues. Mechanisms whereby an RV-specific immune response may develop but immune effectors fail to infiltrate CNS tissues include (i) defects in the proinflammatory response of CNS-resident cells (24
) and (ii) functional changes at the BBB that permit immune cell invasion.
The invasion of infected CNS tissues by immune effector cells from the circulation is dependent upon a sequence of events that includes the production of proinflammatory cytokines and chemokines by CNS-resident cells. These not only attract cells into the tissues but also induce the expression of the adhesion molecules that allow circulating cells to interact with the cells of the BBB. Thus, a reduction in the proinflammatory response may lead to a failure to induce BBB permeability changes and a protective CNS inflammatory response. A recent study has suggested that strains of RV may differ in the ability to induce a CNS proinflammatory response (24
). We expect that the magnitude and kinetics of the innate response to different RV isolates depend upon the ability to spread into and replicate in CNS tissues, as well as, at later stages, to induce immune clearance versus cause disease. Wang et al. found that mRNAs specific for MCP-1, IP-10, RANTES, and a variety of other chemokines and cytokines were strongly elevated in severely paralyzed mice infected with an undisclosed SHBRV isolate but to lesser extents than in similarly afflicted mice infected with the less pathogenic CVS-B2c strain (24
). On the basis of the timing of the development of immunity in CVS-F3-infected mice (21
) and differences in the spread and replication of SHBRV-17 versus CVS-F3, we adjusted our virus inoculums to cause similar virus loads in the CNS tissues as the innate response develops and performed our comparative analyses at day 8 p.i., when the SHBRV-17-infected mice were still healthy. In our comparison of the upregulation of mRNAs specific for proinflammatory markers in the CNS tissues, the responses of SHBRV-17-infected mice were equal to or greater than those of CVS-F3-infected counterparts in the cerebellum, the primary location of the enhanced BBB permeability and CNS inflammation associated with the clearance of CVS-F3 (21
). Consistent with the upregulation of TNF-α and ICAM-1 mRNAs, enhanced ICAM-1 expression can be readily visualized on the neurovasculature of the cerebellum in both CVS-F3- and SHBRV-17-infected mice. This suggests that any activated RV antigen-specific lymphocytes present in the circulation should be capable of adhering to neurovascular endothelial cells.
If the proinflammatory response in the CNS of SHBRV-17-infected mice is intact, the inability of immune cells to infiltrate the cerebellum is more likely to result from a deficit in the mechanism that allows these cells to cross the BBB. Conceivably, this could be at the level of either immune cell or BBB function. We did not detect any reduction in the development of an RV antigen-specific humoral response between SHBRV-17- and CVS-F3-infected mice. Moreover, as has previously been demonstrated for the clearance of CVS-F3 from the CNS, the isotype of the RV-specific antibodies produced by SHBRV-17-infected mice is IgG2a. This indicates a bias toward the Th1 cell reactivity that predominates in other CNS inflammatory reactions (7
). The argument that the cells of the adaptive immune response are fully functional in SHBRV-17-infected mice is supported by the observation that lymphocytes from SHBRV-17-infected mice are capable of enhancing BBB permeability, invading CNS tissues, and clearing CVS-F3 when adoptively transferred into infected rag-2−/−
Although CVS-F3 can be cleared from the CNS by RV-specific lymphocytes raised in either CVS-F3- or SHBRV-17-infected mice, neither effector cell population can clear SHBRV-17 from CNS tissues. The transfer of lymphocytes from SHBRV-17- and CVS-F3-infected mice can protect naive recipients against subsequent infection with SHBRV-17, presumably by preventing the spread of the virus to the CNS. However, we have found that neither cell population can prevent lethal rabies when SHBRV-17 infection precedes adoptive transfer by as little as several hours. In these experiments, antiviral immunity was allowed to develop for 6 days in the donor animals and transferred cells had an additional 7 to 8 days to mediate a protective response before the recipients died. Considering that clearance of CVS-F3 begins approximately 6 days after infection, this should have been sufficient time to mediate a protective response against SHBRV-17. The fact that the course of the disease was unchanged suggests that the adoptively transferred cells had no impact on the replication and spread of the virus in the CNS.
We speculate that the inability to “open” the BBB in the cerebellum and deliver the appropriate immune effectors to the CNS tissues is the fundamental deficit that prevents immune clearance of SHBRV-17 from the CNS and ultimately leads to the death of infected animals. While CD8 T cells are important in clearing some viruses from the CNS, the primary effectors of RV clearance are VNAs (10
) that must either cross the BBB or be produced in the CNS tissues by invading B cells. In our studies of CVS-F3 clearance from CNS tissues, enhanced BBB permeability was associated with invasion of the CNS by CD4 T cells and B cells but not with CD8 T-cell accumulation (21
). Correlations between the patterns of BBB permeability changes and the appearance of CD4 and IFN-γ mRNAs in CNS tissues have led us to speculate that the enhanced BBB permeability associated with the delivery of B cells and antibody to the CNS of CVS-F3-infected mice is driven by CD4 T cells through an IFN-γ-dependent process (21
). The failure of this mechanism could be why conventional postexposure treatment of rabies is ineffective once the virus has reached CNS tissues and clinical signs of the disease have developed (3
). We suggest that at some time after the virus has reached the CNS tissues, the BBB in the cerebellum becomes refractory to the signals that trigger enhanced permeability. Consequently, immune effectors cannot reach the CNS tissues to clear the virus. This is consistent with observations made in clinical rabies that people who die from rabies in the absence of treatment often develop RV-specific antibodies (15
). The idea that the development of enhanced BBB permeability is a critical checkpoint in the response to RV infection of the CNS is also supported by a recent human rabies case where an individual who did not receive PEP recovered despite developing advanced clinical signs of infection (5
). In this case, the victim naturally developed a high VNA titer and enhanced BBB permeability as evidenced by the appearance of serum proteins in the cerebrospinal fluid (26
). Even in the absence of PEP, the indigenous immune response to RV in this individual was sufficient to clear the infection from the CNS. Since RV can be cleared from the CNS if immune effectors have access to the infected tissues, we believe that the development of an approach to circumvent the maintenance of BBB integrity in RV-infected individuals may have therapeutic value.
The existence of a mechanism to maintain BBB integrity and prevent the delivery of immune effectors to CNS tissues has implications for a variety of CNS diseases. It is conceivable that this mechanism interferes with the clearance of other neurotropic virus and possibly with CNS tumor immunity. From the opposite perspective, the ability to trigger such a mechanism may have therapeutic value for neurodegenerative diseases associated with a reduction of BBB integrity.