Recombinant myelin oligodendrocyte glycoprotein quality modifies evolution of experimental autoimmune encephalitis in macaques.

Experimental autoimmune encephalitis (EAE) is a well-recognized model for the study of human acquired demyelinating diseases (ADD), a group of inflammatory disorders of the central nervous system (CNS) characterized by inflammation, myelin loss, and neurological impairment of variable severity. In rodents, EAE is typically induced by active immunization with a combination of myelin-derived antigen and a strong adjuvant as complete Freund's adjuvant (CFA), containing components of the mycobacterial wall, while myelin antigen alone or associated with other bacterial components, as lipopolysaccharides (LPS), often fails to induce EAE. In contrast to this, EAE can be efficiently induced in non-human primates by immunization with the recombinant human myelin oligodendrocyte glycoprotein (rhMOG), produced in Escherichia coli (E. coli), purified and formulated with incomplete Freund's adjuvant (IFA), which lacks bacterial elements. Here, we provide evidence indicating how trace amounts of bacterial contaminants within rhMOG may influence the course and severity of EAE in the cynomolgus macaque immunized with rhMOG/IFA. The residual amount of E. coli contaminants, as detected with mass spectrometry within rhMOG protein stocks, were found to significantly modulate the severity of clinical, radiological, and histologic hallmarks of EAE in macaques. Indeed, animals receiving the purest rhMOG showed milder disease severity, increased numbers of remissions, and reduced brain damage. Histologically, these animals presented a wider diversity of lesion types, including changes in normal-appearing white matter and prephagocytic lesions. Non-human primates EAE model with milder histologic lesions reflect more accurately ADD and permits to study of the pathogenesis of disease initiation and progression.

Recovery and quantification of a myelin oligodendrocyte glycoprotein peptide from rat plasma after protein precipitation.

The recovery of high molecular weight peptides from complex biological samples is a challenging task. Herein, a reliable, cost effective and rapid methodology was developed for the recovery and quantification of a myelin oligodendrocyte glycoprotein epitope namely (LysGly)5MOG35-55, from rat plasma. Removal of plasma proteins before quantification of the peptide was achieved after precipitation by an acetonitrile/water/formic acid solution. Using the developed protocol, average recoveries of the peptide from plasma ranged between 83.3 and 90.3%.

Effects of peptide fraction and counter ion on the development of clinical signs in experimental autoimmune encephalomyelitis.

The most commonly used immunogen to induce experimental autoimmune encephalomyelitis is MOG35-55 , a 21-residue peptide derived from myelin oligodendrocyte glycoprotein (MOG). In most studies, mice exhibit a chronic disease; however, in some studies mice show a transient disease. One variable that is not often controlled for is the peptide fraction of the purified MOG material, which can vary from less than 50% to over 90%, with the remainder of mass primarily comprised of the counter ion used for peptide purification. We compared the development of clinical signs in female C57Bl6 mice immunized with two commercially available MOG35-55 peptides of similar purity but different peptide fraction (MOG-A being 45%; MOG-B being 72%). A single immunization with MOG-A induced a chronic disease course with some recovery at later stages, whereas immunization with MOG-B induced a similar course of disease but with significantly lower average clinical scores despite a higher peptide content. The addition of a booster immunization significantly increased clinical severity with both preparations, and significantly reduced the average day of onset using MOG-A. To determine if the counter ion could influence disease, we compared MOG-B-containing trifluoroacetate with MOG-B-containing acetate. Although disease incidence and severity were similar, the average day of disease onset occurred approximately 5 days earlier with the use of MOG-B-containing trifluoroacetate. These results demonstrate that differences in peptide fraction influence the course of encephalomyelitis disease, which may be due in part to the levels of counter ions present in the purified material. These findings underscore the fact that a knowledge of peptide fraction is as critical as knowledge of peptide purity when using peptides from different sources.