Germ Line IgM Is Sufficient, but Not Required, for Antibody-Mediated Alphavirus Clearance from the Central Nervous System

Abstract

Sindbis virus (SINV) infection of neurons in the brain and spinal cord in mice provides a model system for investigating recovery from encephalomyelitis and antibody-mediated clearance of virus from the central nervous system (CNS). To determine the roles of IgM and IgG in recovery, we compared the responses of immunoglobulin-deficient activation-induced adenosine deaminase-deficient (AID), secretory IgM-deficient (sIgM), and AID sIgM double-knockout (DKO) mice with those of wild-type (WT) C57BL/6 mice for disease, clearance of infectious virus and viral RNA from brain and spinal cord, antibody responses, and B cell infiltration into the CNS. Because AID is essential for immunoglobulin class switch recombination and somatic hypermutation, AID mice produce only germ line IgM, while sIgM mice secrete IgG but no IgM and DKO mice produce no secreted immunoglobulin. After intracerebral infection with the TE strain of SINV, most mice recovered. Development of neurologic disease occurred slightly later in sIgM mice, but disease severity, weight loss, and survival were similar between the groups. AID mice produced high levels of SINV-specific IgM, while sIgM mice produced no IgM and high levels of IgG2a compared to WT mice. All mice cleared infectious virus from the spinal cord, but DKO mice failed to clear infectious virus from brain and had higher levels of viral RNA in the CNS late after infection. The numbers of infected cells and the amount of cell death in brain were comparable. We conclude that antibody is required and that either germ line IgM or IgG is sufficient for clearance of virus from the CNS. Mosquito-borne alphaviruses that infect neurons can cause fatal encephalomyelitis. Recovery requires a mechanism for the immune system to clear virus from infected neurons without harming the infected cells. Antiviral antibody has previously been shown to be a noncytolytic means for alphavirus clearance. Antibody-secreting cells enter the nervous system after infection and produce antiviral IgM before IgG. Clinical studies of human viral encephalomyelitis suggest that prompt production of IgM is associated with recovery, but it was not known whether IgM is effective for clearance. Our studies used mice deficient in production of IgM, IgG, or both to characterize the antibody necessary for alphavirus clearance. All mice developed similar signs of neurologic disease and recovered from infection. Antibody was necessary for virus clearance from the brain, and either early germ line IgM or IgG was sufficient. These studies support the clinical observation that prompt production of antiviral antibody is a determinant of outcome.

Both ADP-Ribosyl-Binding and Hydrolase Activities of the Alphavirus nsP3 Macrodomain Affect Neurovirulence in Mice

“…Enzyme immunoassays. SINV-specific antibody in serum and 10% (wt/vol) brain and spinal cord homogenates was measured using an in-house EIA (89). Maxisorp 96-well plates (Thermo Scientific Nunc) were coated with 10 6 PFU/well of SINV strain TE in 50 mM NaHCO 3 (pH 9.6) at 4°C overnight.…”

Interferon regulatory factors 3 and 7 have distinct roles in the pathogenesis of alphavirus encephalomyelitis

“…Effect of IRF3 or IRF7 deficiency on the production of SINV-specific antibody Because virus-specific antibody produced within the CNS is important for virus control and clearance [33,34], we examined the levels of SINV-specific IgM ( Fig. 3c) and IgG ( Fig.…”

“…An in-house EIA for SINV-specific IgM and IgG was performed as previously described [33]. Briefly, Maxisorp (Nunc) 96-well plates were coated with 10 6 p.f.u.…”

Genetic control of alphavirus pathogenesis

“…Ifng− / − and Ifngr1− / − mice also have significantly fewer B cells and lower mRNA expression levels of B cell attracting chemokines, including Cxcl9, Cxcl10 , and Cxcl13 , compared to WT mice, suggesting cooperative virus clearance between IFN-γ and anti-SINV antibody occurs via IFN-γ promotion of antibody-secreting B cell chemotaxis into the CNS (Baxter and Griffin 2016 ). Germline IgM or IgG alone is sufficient for SINV clearance from the CNS, with AID − / − mice (only produce IgM) and sIgM − / − mice (secrete IgG but not IgM) showing comparable morbidity and virus clearance compared to WT mice (Nilaratanakul et al 2018 ). In contrast, AID − / − sIgM − / − double knockout mice are unable to clear infectious virus from the brain and show persistently high viral RNA levels, further supporting the important role of anti-SINV antibody in virus clearance (Nilaratanakul et al 2018 ).…”

“…Germline IgM or IgG alone is sufficient for SINV clearance from the CNS, with AID − / − mice (only produce IgM) and sIgM − / − mice (secrete IgG but not IgM) showing comparable morbidity and virus clearance compared to WT mice (Nilaratanakul et al 2018 ). In contrast, AID − / − sIgM − / − double knockout mice are unable to clear infectious virus from the brain and show persistently high viral RNA levels, further supporting the important role of anti-SINV antibody in virus clearance (Nilaratanakul et al 2018 ). Further studies examining the exact mechanism by which anti-SINV antibody clears infectious virus from neurons in a noncytolytic manner are underway.…”