Previous studies have demonstrated that the chimeric TBEV/DEN4Δ30 virus was highly attenuated for neuroinvasiveness in immunodeficient mice (39
) and was less neurovirulent in adult Swiss mice than its immediate TBEV/DEN4 parent or LGTV (Pletnev, unpublished data). In addition, TBEV/DEN4Δ30 virus was shown to induce high levels of serum neutralizing antibody against TBEV in mice and rhesus monkeys and provided complete protection against challenge with TBEV or LGTV (30
). Nevertheless, TBEV/DEN4Δ30 retained a high level of neurovirulence in suckling mice inoculated i.c. (39
). The relevance of flavivirus-induced death in suckling mice to the potential to cause CNS disease in humans is unclear since death in suckling mice is common even after i.c. inoculation of highly attenuated flaviviruses, including live attenuated YF 17D vaccine (23
), which has a remarkable record of safety and efficacy in humans (21
). Nonetheless, we decided to evaluate the neurovirulence of TBEV/DEN4Δ30 in nonhuman primates, especially since the chimeric virus carries the structural prM and E protein genes derived from the highly neurovirulent Sofjin strain of Far Eastern TBEV. In contrast to TBEV/DEN4Δ30, chimeric vaccine candidates for West Nile virus (WNV/DEN4Δ30) and LGTV (LGT/DEN4) have entered clinical trials in humans without monkey neurovirulence testing, since both vaccine candidates were highly attenuated for the CNS of suckling mice (35
). Results of the phase I clinical trials in humans indicate that both vaccines are immunogenic and safe in adult volunteers without local or systemic reactogenicity. Unfortunately, seroconversion against heterologous TBEV in the LGT/DEN4 vaccine recipients was infrequent, and the level of TBEV-neutralizing antibodies induced was significantly lower than that observed against homologous LGTV, suggesting that the vaccine candidate will need to be based on the TBEV structural protein genes (48
Several factors need to be taken into account during the evaluation of the level of neurovirulence of TBEV/DEN4Δ30 in nonhuman primates. First, although the MNVT has been developed for a variety of neurotropic viruses, including poliovirus (25
), measles virus (40
), mumps virus (14
), and vesicular stomatitis virus (10
), the MNVT has been established for only one flavivirus, the YF 17D vaccine virus (13
). Second, a decision regarding an acceptable level of neurovirulence for a test virus should be made based on a comparison to one or more reference control viruses, which usually include the neurovirulent parent virus and a related vaccine virus that exhibits an acceptable level of attenuation for the CNS of humans. The YF 17D vaccine virus can be used as a surrogate, and the naturally attenuated LGTV that retains an unacceptable level of residual neurovirulence for humans (41
) acts as the surrogate for the TBEV parent, which is a BSL-4 agent. Third, the histopathological scoring methodology for the evaluation of YF 17D vaccine might not be appropriate for the assessment of neurovirulence of the antigenically distant TBEV/DEN4Δ30. Finally, the standard MNVT provides only a snapshot of the pathogenesis of CNS infection, usually around 30 days after i.c. inoculation of monkeys with a given virus, and does not include evaluation of the time course of either viral replication or the development of histopathological lesions in the CNS. Thus, our major objective was to study neuropathogenesis in rhesus monkeys following i.c. inoculation with TBEV/DEN4Δ30 virus and to compare the level of neurovirulence of the chimeric vaccine candidate with that of LGTV or YF 17D vaccine virus in order to gather comprehensive safety data prior to advancing to clinical trials.
TBEV can cause acute, subacute, or chronic forms of encephalitis in humans and experimental monkeys (2
). In this study, the clinical data indicate that TBEV/DEN4Δ30 virus retains significant neurovirulence. Although the majority of animals inoculated with TBEV/DEN4Δ30 showed only moderate signs of neurological illness, there was one monkey that developed severe encephalitis. However, the TBEV/DEN4Δ30 virus is significantly attenuated compared to the highly virulent parental TBEV, since it has been previously shown that i.c. inoculation of monkeys with this strain resulted in the development of seizures, tremors, pareses, paralysis, and death in 100% of animals (37
). Among LGTV-inoculated monkeys, one animal developed severe encephalitis, but the remaining monkeys had tremors and limb weakness consistent with those previously described (24
). It is noteworthy that YF 17D, a safe vaccine in humans (21
), can cause signs of CNS dysfunction such as tremors in 40% of i.c. inoculated monkeys (22
). The clinical data for monkeys therefore suggested that the TBEV/DEN4Δ30 virus was insufficiently attenuated for the CNS of nonhuman primates.
Although the neurovirulence of selected flaviviruses in nonhuman primates has been examined previously (11
), the present study is the first to analyze the kinetics of replication of attenuated flaviviruses in multiple anatomic sites of the CNS of monkeys. The neurons, as in many flaviviral infections of the CNS (4
), were the primary cells expressing virus antigen. The anatomical distribution of infected neurons in the CNS of monkeys inoculated with each of the three attenuated flaviviruses correlated with the sites of virus recovery. The viral loads in the CNS of the majority of monkeys inoculated with attenuated flaviviruses in this study were relatively low (≤3.3 log10
PFU/g) compared to high titers (>6 log10
PFU/g) produced by virulent strains of TBEV (11
). These virological data, like the clinical observations summarized above, indicate that the TBEV/DEN4Δ30 vaccine candidate is highly attenuated compared to its parental TBEV. Another indication of the attenuation of TBEV/DEN4Δ30 was its failure to cause viremia in i.c. inoculated monkeys whereas either LGTV or YF 17D was detected in the blood. Detectable replication of infectious virus in the CNS of the majority of monkeys inoculated with TBEV/DEN4Δ30 or YF 17D ceased after day 7, coinciding with the appearance of serum neutralizing antibody. In contrast, the humoral immune response did not appear to have a significant role in the clearance of LGTV from the CNS, since its replication and caudal spread continued until day 21 despite the high titers of serum neutralizing antibodies developed at earlier time points (days 6 to 8). Although the levels of replication of TBEV/DEN4Δ30 and YF 17D virus in the CNS were generally similar, one TBEV/DEN4Δ30-inoculated monkey with severe encephalitis demonstrated high virus loads throughout the entire CNS. The virus recovered from the brain or spinal cord of this monkey was genetically identical to the inoculated TBEV/DEN4Δ30 virus, indicating that host factors, rather than mutations, might be responsible for the increased viral burden in the CNS and clinical manifestations of encephalitis.
Histopathological analysis of the CNS of rhesus monkeys inoculated with TBEV/DEN4Δ30, LGTV, or YF 17D revealed typical, although not disease-specific, histopathological lesions consistent with those previously described for flaviviral encephalitides in humans (7
) and in experimentally infected monkeys (2
). The spectrum of virus-associated histopathology was comprised of features characteristic of two pathological processes: infiltration by inflammatory cells and reaction of the resident cells of the CNS (MGA/ND). In this study, we developed a new histopathological scoring methodology that included a separate evaluation of these two processes, and we evaluated all major neuroanatomical compartments (cerebral cortex, hippocampus, basal ganglia, thalamus, midbrain, pons, cerebellum, medulla oblongata, and spinal cord). The histopathological analysis revealed that the mean group scores for the entire CNS were significantly higher for monkeys inoculated with TBEV/DEN4Δ30 than for monkeys inoculated with either LGTV or YF 17D at all time points starting from day 7. Thus, the results of histopathological analysis of the CNS together with clinical observations and data on virus replication strongly indicate that chimeric TBEV/DEN4Δ30 virus is not sufficiently attenuated for the CNS of nonhuman primates. It is interesting that extensive histopathology in the CNS was observed with a relatively low level of TBEV/DEN4Δ30 replication, indicating that the virus evokes a strong cellular inflammatory response. In addition, using a new histopathological scoring methodology, we were able to identify distinct differences in the neuropathogenesis induced by TBEV/DEN4Δ30, LGTV, or YF 17D. For example, LGTV induced more severe lesions in the brain stem, cerebellum, and spinal cord, which became striking on day 21. These findings are consistent with previously described neurovirulence scores for LGTV, which were low in the hemispheres, intermediate in the brain stem, and high in the spinal cord (24
). In contrast, the TBEV/DEN4Δ30 and YF 17D viruses demonstrated remarkably similar spatiotemporal histopathological profiles, which were high in the hemispheres with a progressive decrease toward the spinal cord. The anatomical compartments affected in monkeys inoculated with YF 17D in our study were similar to the previously described “indicator centers” (24
) or “target and discriminator areas” (13
) except that our finding of extensive involvement of the cerebral cortex is in disagreement with previous conclusions that this major CNS compartment can be designated a “spared area” (13
). It should be noted here that the differences in anatomical distribution of histopathological lesions induced by each of these three viruses became less evident at 30 dpi, suggesting that histopathological analysis of the CNS in the MNVT should not be limited to only this time point.
The fact that YF 17D, LGTV and TBEV/DEN4Δ30 are antigenically distant flaviviruses might partially explain the observed differences in spatiotemporal profiles of viral replication and virus-associated histopathology in the CNS. The structural E protein of flaviviruses is involved in virus entry into cells and virion assembly and maturation, and it is believed to be a major determinant of virulence (21
). The E protein of chimeric TBEV/DEN4Δ30 virus is derived from a highly virulent Far Eastern TBEV strain and shares amino acid homology of only 42% with that of YF 17D (34
) and 88% with that of LGTV (16
). Since TBEV/DEN4Δ30 contains the capsid and nonstructural protein genes of the nonneuroinvasive and low neurovirulent DEN4 virus, it is tempting to speculate that the prM and E proteins of TBEV, rather than the DEN4 genes, are the genetic determinants that are primarily responsible for the extensive histopathology in the CNS, but this will require additional studies in the future. Clearly, the differences in the biology of viruses and in their tropism to various neuronal populations within the CNS make direct comparisons of the levels of neurovirulence of different attenuated flaviviruses very difficult.
The pathogenesis of neurotropic flavivirus infections involves complex virus-host interactions, with a number of factors that have an effect on virus replication and its clearance by the innate and adaptive immune responses induced both in the periphery and within the CNS. Pathogenesis studies with nonhuman primates have been limited to quantitating the peripheral immune responses or the disease burden caused by replication of virus in the CNS (4
). To the best of our knowledge, this is the first study with nonhuman primates to analyze the phenotype of cells involved in the inflammatory response within the CNS following i.c. inoculation with attenuated flaviviruses. We observed vigorous MGA in the areas of neuronal damage. The degenerating neurons were often found engulfed by the CD68+
activated microglia/macrophages. We show that inflammatory foci were composed of CD3+
T lymphocytes, CD8+
B cells, and CD68+
macrophages/microglia. Numerous CD3+
T lymphocytes were present in perivascular and parenchymal compartments of the brains and spinal cords of infected monkeys. However, only a small fraction of CD3+
T lymphocytes was represented by CD8+
CTLs within the perivascular compartments, whereas the parenchyma was infiltrated mostly by CD8+
CTLs. Importantly, CD8+
CTLs were often found in the immediate vicinity of the remnants of degenerated neurons and within the MGNs but also in close contact with morphologically intact neurons, suggesting that, while exhibiting their effector function in the clearance of neuroinfection, these cells might play a role in collateral neuronal damage. CD20+
B cells were also found within the perivascular inflammatory infiltrates, but numerous B cells were recruited into the surrounding neuropil, suggesting their involvement in virus clearance by local production of neutralizing antibodies. Further studies using quantitative immunohistochemical analysis are needed to determine whether the magnitude and dynamics of the cellular inflammatory response within the CNS are influenced by the level of neurovirulence of the virus and how these responses contribute to viral clearance and potential bystander cell injury.
Overall, the results of this study indicate that chimerization of a highly neurovirulent strain of TBEV with nonneuroinvasive DEN4 in the presence of the Δ30 mutation had an attenuating effect on neurovirulence in nonhuman primates compared to parental TBEV. However, this effect was insufficient based on comparison with two other attenuated flaviviruses, LGTV and YF 17D. Further reduction in the level of neurovirulence of the chimeric TBEV/DEN4Δ30 virus is needed. Nevertheless, the facts that the histopathological profile within the CNS of TBEV/DEN4Δ30-inoculated monkeys was remarkably similar to that observed in monkeys inoculated with YF 17D vaccine virus and that TBEV/DEN4Δ30 seems to have a reduced ability to affect the motor neurons of the spinal cord compared to LGTV or highly virulent TBEV strains (6
) suggest that chimerization of TBEV with DEN4Δ30 is a useful mechanism for attenuation. An interesting aspect of this study is the comparative virological and histopathological analysis of the evolution of CNS infection in nonhuman primates following i.c. inoculation of attenuated flaviviruses. Our results provide insight into the neuropathogenesis beyond the most often analyzed time point at 30 dpi and demonstrate that attenuated flaviviruses can be more reliably discriminated by their histopathological profile at earlier time points. Taken together, these findings and our modified methodology of histopathological evaluation of neurovirulence may guide the design of safe live vaccines against neurotropic flaviviruses. Additional quantitative analysis is needed to evaluate the dynamics of CNS infiltration by different populations of inflammatory cells and responses of resident cells of the CNS induced by attenuated flaviviruses. We are currently performing a computerized morphometric analysis of the cellular inflammatory responses within the CNS of monkeys to investigate their role in neuropathogenesis and contribution to the outcome of neuroinfection.