Herpesvirus trigger accelerates neuroinflammation in a nonhuman primate model of multiple sclerosis

Abstract

Pathogens, particularly human herpesviruses (HHVs), are implicated as triggers of disease onset/progression in multiple sclerosis (MS) and other neuroinflammatory disorders. However, the time between viral acquisition in childhood and disease onset in adulthood complicates the study of this association. Using nonhuman primates, we demonstrate that intranasal inoculations with HHV-6A and HHV-6B accelerate an MS-like neuroinflammatory disease, experimental autoimmune encephalomyelitis (EAE). Although animals inoculated intranasally with HHV-6 (virus/EAE marmosets) were asymptomatic, they exhibited significantly accelerated clinical EAE compared with control animals. Expansion of a proinflammatory CD8 subset correlated with post-EAE survival in virus/EAE marmosets, suggesting that a peripheral (viral?) antigen-driven expansion may have occurred post-EAE induction. HHV-6 viral antigen in virus/EAE marmosets was markedly elevated and concentrated in brain lesions, similar to previously reported localizations of HHV-6 in MS brain lesions. Collectively, we demonstrate that asymptomatic intranasal viral acquisition accelerates subsequent neuroinflammation in a nonhuman primate model of MS.

Keywords: EAE; human herpesvirus 6; marmoset; multiple sclerosis; viral trigger.

Fig. 1.

HHV-6 viral antigen occasionally detected in the brain after intranasal inoculations. Two of nine examined brain regions from HHV-6B–inoculated marmoset M26 contained detectable HHV-6 late antigen p101k (red; arrows), with cytoplasmic localization. (A) Olfactory bulb. (B) Hippocampus. (Scale bar: 50 µm; Inset scale bar: 5 µm.)

Fig. 2.

Significantly accelerated clinical EAE in HHV-6 intranasally inoculated marmosets. (A) Reduced time to symptom onset in virus/EAE marmosets compared with SupT1 control/EAE marmosets (unpaired t test, P = 0.04). (B) Shortened survival post-EAE induction in virus/EAE marmosets compared with SupT1 control/EAE marmosets (unpaired t test, P = 0.03).

Fig. 3.

Characterization of EAE brain lesions. PD (A) and T1 (B) in vivo images from HHV-6B/EAE marmoset M28. Two white matter lesions are numbered. (C) Final PD and T1 lesion volumes by marmoset for all groups. (D and E) Virus/EAE marmosets survived for less time after the onset of PD (unpaired t test, P = 0.04) (D) and T1 (unpaired t test, P = 0.03) (E) brain lesions. (F) HHV-6B/EAE marmosets had the greatest T1 lesion burden.

Fig. 4.

HHV-6 viral antigen is up-regulated in virus/EAE marmoset brains. Arrows indicate positivity for HHV-6 late antigen p101k (red) in inflamed brain regions from representative virus/EAE marmoset M28. (A and B) Temporal white matter around putamen. (C) Leptomeninges around hippocampal fissure. (Scale bar: 50 µm.)

Fig. 5.

Characterization of HHV-6–positive brain lesions from a virus/EAE marmoset. (A) Serial sections of an HHV-6–positive perivascular EAE lesion demonstrate CD3 T and CD20 B cell infiltrates, MRP14⁺ macrophages/monocytes, Iba-1⁺ microglia, blood-brain barrier leakiness evidenced by fibrinogen deposition, and demyelination evidenced by the absence of myelin PLP. (Scale bar: 100 µm.) (B) HHV-6 antigen (brown) colocalizes with CD3 T cells (blue). (C) HHV-6–infected cells in green (Alexa 488). (D) CD3 T cells in red (Alexa 594), with DAPI counterstain (gray). (E) Merged image with arrows showing colocalization of HHV-6 viral antigen with CD3 T cells. (Scale bar: B–E, 50 µm.) The lesion pictured in A is located in the gray matter (lateral dorsal thalamic nucleus), whereas the lesion pictured in B–E is located in the temporal white matter. Lesions are from HHV-6B/EAE marmoset M28.

Fig. 6.

Increase in IFN-γ–producing effector/memory (E/M) CD8 T cells significantly correlates with disease duration in virus-inoculated marmosets. (A) Representative flow cytometry plot of CD8⁺ CD45RA/CD27 T cell subsets, with histogram showing highest IFN-γ production by E/M subset. E, effector; M, memory. (B) Representative flow cytometry plots showing a decrease in naïve CD8⁺ T cells at terminal EAE time points in virus/EAE, but not control/EAE, marmosets. (C and D) Contraction of naïve CD8 (C) and expansion of E/M CD8 (D) T cells correlate with EAE disease duration in virus-inoculated marmosets. (E and F) In the control/EAE marmosets, no such correlation was observed for either the naïve CD8 (E) or the E/M CD8 (F) subset.