Comparative neuropathogenesis and neurovirulence of attenuated flaviviruses in nonhuman primates

Abstract

Based on previous preclinical evaluation in mice and monkeys, the chimeric TBEV/DEN4Delta30 virus, carrying the prM and E protein genes from a highly virulent Far Eastern strain of tick-borne encephalitis virus (TBEV) on the backbone of a nonneuroinvasive dengue type 4 virus (DEN4), has been identified as a promising live attenuated virus vaccine candidate against disease caused by TBEV. However, prior to use of this vaccine candidate in humans, its neurovirulence in nonhuman primates needed to be evaluated. In the present study, we compared the neuropathogeneses of the chimeric TBEV/DEN4Delta30 virus; Langat virus (LGTV), a former live TBEV vaccine; and yellow fever 17D virus vaccine (YF 17D) in rhesus monkeys inoculated intracerebrally. TBEV/DEN4Delta30 and YF 17D demonstrated remarkably similar spatiotemporal profiles of virus replication and virus-associated histopathology in the central nervous system (CNS) that were high in cerebral hemispheres but progressively decreased toward the spinal cord. In contrast, the neurovirulence of LGTV exhibited the reverse profile, progressing from the site of inoculation toward the cerebellum and spinal cord. Analysis of the spatiotemporal distribution of viral antigens in the CNS of monkeys revealed a prominent neurotropism associated with all three attenuated viruses. Nevertheless, TBEV/DEN4Delta30 virus exhibited higher neurovirulence in monkeys than either LGTV or YF 17D, suggesting insufficient attenuation. These results provide insight into the neuropathogenesis associated with attenuated flaviviruses that may guide the design of safe vaccines.

FIG. 1.

Distribution of daily clinical scores in monkeys following i.t. inoculation with TBEV/DEN4Δ30 (A) or LGTV (B) in experiment 1. There were 10 animals inoculated with each virus. Since 1 of the 10 animals was excluded from the TBEV/DEN4Δ30 group (see Results), the daily clinical scores are shown in panel A for only 9 monkeys.

FIG. 2.

Viremia (bars) and serum neutralizing antibody response to homologous virus (lines) in monkeys following i.t. inoculation with TBEV/DEN4Δ30, LGTV, or YF 17D. The dashed line shows the lowest serum dilution tested (1:2) by PRNT₆₀. The mean virus titers in sera of monkeys inoculated with LGTV or YF 17D are shown by black or open bars, respectively. Values below the limit of virus detection in serum (0.7 log₁₀ PFU/ml) are not shown.

FIG. 3.

Viral loads in the CNS of monkeys following i.t. inoculation with TBEV/DEN4Δ30 (A), LGTV (B), or YF 17D (C) on the indicated dpi (experiments 2 and 3). The virus titers in CNS regions of each monkey were measured by plaque-forming assay as described in Materials and Methods and are expressed as means and standard deviations. The lower limit of virus detection was 1.7 log₁₀ PFU/g of brain or spinal cord tissue (values below the detection limit are not shown). Gray bars show viral loads in CNS regions of monkeys inoculated with TBEV/DEN4Δ30 or LGTV in experiment 3 (monkeys are indicated with asterisks).

FIG. 4.

Spectrum of virus-associated histopathological lesions in the CNS of monkeys inoculated with TBEV/DEN4Δ30 (A, D, and G), LGTV (B, E, and H), or YF 17D (C, F, and I) at the peak time points (14 to 21 dpi). (A to C) Frontal cortex (21 dpi), showing perivascular cuffing by mononuclear inflammatory cells (black arrows) and (A) diffuse mononuclear inflammatory cell infiltration in the neuropil (white arrow), focal gliosis, and ND (arrowhead); (B) MGN (arrowhead); and (C) focal gliosis and neuronophagia (arrowhead). (D to F) Cerebellum (14 dpi), showing CII in the leptomeninges (black arrows), focal gliosis in the molecular layer (white arrows), and degeneration and loss of Purkinje cells (arrowheads). (G to I) Ventral horns of the cervical (G) and lumbar (H and I) spinal cord (14 to 21 dpi), showing (G) neuronophagia and acidophilic neuronal necrosis (arrowhead, 21 dpi), (H) neuronophagia and degenerating eosinophilic neuron with diffuse chromatolysis and eccentric nucleus (arrowhead, 14 dpi), and (I) foci of neuronophagia (arrowheads, 14 dpi). Hematoxylin and eosin staining was used. Original magnifications, ×200 (A to F, H, and I) and ×400 (G).

FIG. 5.

Temporal and anatomical distributions of histopathological lesions in the CNS of monkeys following i.t. inoculation with TBEV/DEN4Δ30, LGTV, or YF 17D. (A) Temporal distributions of MGHS for the CII and MGA/ND in the entire CNS. The grading scale is described in Table S1 in the supplemental material. (B and C) Anatomical profiles of the CII (B) and MGA/ND (C) at 14, 21, and 30 dpi. MGHS did not differ statistically unless specifically noted. MGHS were significantly higher (▴) or lower (▾) compared to the previous time point (P < 0.05). MGHS for the CNS regions of the TBEV/DEN4Δ30 group of monkeys were significantly higher (P < 0.05) than those for the LGTV group (indicated with one asterisk) or the YF 17D group (indicated with two asterisks) at the same time point. MGHS for the spinal cords of LGTV-inoculated monkeys were significantly higher (P < 0.05) than those for the TBEV/DEN4Δ30 or YF 17D group on 21 dpi (indicated with three asterisks). Hemispheres: cortex, hippocampus, basal ganglia (caudate nucleus, globus pallidus, and putamen), and thalamus. Brain stem: midbrain, pons, and medulla oblongata. Spinal cord: cervical, thoracic, and lumbar regions.

FIG. 6.

Localization of viral antigens in the CNS of monkeys inoculated with TBEV/DEN4Δ30 at 7 dpi (A and B), with LGTV at 14 dpi (C and D), or with YF 17D at 7 dpi (E and F). (A) Frontal cortex, showing strongly immunolabeled neurons (arrows) within intact gray matter. (B) Thalamus, showing immunolabeled neurons (arrows) surrounding a perivascular inflammatory infiltrate (arrowhead). (C and D) Ventral horns of the lumbar spinal cord, showing neuronophagia (D) and prominent immunolabeling in the cytoplasm of degenerating neurons (C and D, arrows). (E) Parietal cortex, showing neuronophagia (arrow) and strongly immunolabeled neurons with decorated processes. (F) Putamen, showing CII (arrowhead) and prominent immunolabeling of neuronal network (arrows). Original magnifications, ×400 (A to E) and ×200 (F).

FIG. 7.

Immunohistochemical analysis of cellular inflammatory response in the CNS. Results of immunostaining for CD3 (A to C), CD8 (D to F), CD20 (G to I), and CD68 (J to L) of mock-inoculated control monkeys (A, D, G, and J; representative sections of caudate nucleus) and monkeys inoculated with TBEV/DEN4Δ30 (B, E, H, and K; adjacent sections of the caudate nucleus) or LGTV (C, F, I, and L; representative sections showing the ventral horn of the lumbar spinal cord) at 21 dpi are shown. (A) A few CD3⁺ T lymphocytes adjacent to endothelial cells of the blood vessel (arrow) or in the surrounding parenchyma (arrowhead). (B) CD3⁺ T lymphocytes represent a fraction of mononuclear cells within the perivascular inflammatory infiltrate (arrow) and diffusely infiltrate the surrounding parenchyma (arrowhead). (C) Diffuse infiltration by CD3⁺ T lymphocytes. Note that many CD3⁺ T lymphocytes are located in close contact with neurons (arrows). (D) No CD8 immunostaining. (E) CD8⁺ CTLs represent only a small fraction of CD3⁺ T lymphocytes within the perivascular compartment (arrow) but make up most of the CD3⁺ T lymphocytes present in the parenchymal compartment (arrowhead). (F) Many CTLs are located in a close contact with neurons (black arrowheads) or in the immediate vicinity of the remnants of neurons (white arrowheads), as well as within the MGN (arrow). (G) Single CD20⁺ B cell within the lumen of the blood vessel (arrow). (H and I) CD20⁺ B cells represent a large fraction of lymphocytes within the perivascular infiltrates (arrows) and also invade the surrounding parenchyma (arrowheads). (J) A few CD68⁺ perivascular macrophages (arrow) and microglial cells (arrowhead). (K) CD68⁺ macrophages within the perivascular inflammatory infiltrates (arrow) and CD68⁺ immunoreactive activated microglia (arrowhead). (L) CD68⁺ activated microglia within the foci of neuronophagia (arrows). Original magnification, ×200.