Booster immunizations with IgV-MOG in IFA caused a chronic course of atypical EAE.

<p>Seven Lewis rats were immunized with a mixture of 25 µg of GP69-88 and 50 µg of rat IgV-MOG in CFA on day 0. After resolution of a classical monophasic bout of EAE, the same rats were boosted with 200 µg of rat IgV-MOG in IFA on days 45 and 85. Rats were scored daily for clinical signs of EAE. After the IgV-MOG boosts in IFA, the incidence of chronic atypical EAE was 100% (7 of 7 rats). The red arrows (bottom panel) mark the dates of the boosts. Shown are the individual disease courses for the 7 rats. The blue line divides the clinical data into classical (left) and atypical (right) EAE courses.</p

The isotype of the macaca anti-MOG Ab was IgG.

<p>Shown are GFP⁻ non-transfected HEK (A) or GFP⁺ macaca MOG HEK (B–F) that were stained with macaca serum collected on days 0, 22, 36, 49, or 63 (A–E) and 93, 210, 469, or 730 (F) post-immunization as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110048#pone-0110048-t001" target="_blank">Table 1</a>. HEK were subsequently stained with an allophycocyanin-conjugated secondary Ab specific for macaca Ab isotypes, including anti-IgG/M/A (A–B), anti-IgG (C, F), anti-IgM (D), anti-IgA (E).</p

Lewis rats immunized with IgV-MOG developed Ab specific for conformational epitopes of MOG.

<p>(A) Lewis rats were immunized with IgV-MOG in CFA, boosted with IgV-MOG in IFA, and sera were taken on day 75. Clinical courses of donor rats are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110048#pone-0110048-g001" target="_blank">Figure 1</a> (numbered 1–7 top to bottom). Shown are GFP⁺-gated rat MOG HEK cells stained with: Black trace = normal rat serum; Blue trace = rat #2 with clinical score of 0; Red trace = rat #1 with clinical score of 1.0; Green trace = rat #3 with score of 3.0. (B) Lewis rats were immunized with IgV-MOG in IFA via the same antigens and schedule as designated for the NHP in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110048#pone-0110048-t001" target="_blank">Table 1</a>. These rats (n = 3) did not exhibit clinically evident EAE. Shown are histograms of GFP⁺-gated rat MOG HEK cells labeled with normal rat serum (black trace) or immune serum from two separate asymptomatic rats (red versus blue) taken 442, 116, 416, and 93 days (top to bottom) after the day of immunization (DOI). Shaded traces indicate at least one year since DOI. Labeled cells were detected with an allophycocyanin-conjugated secondary goat-anti-rat IgG/M secondary Ab. Analyses were of individual serum samples, not pooled sera.</p

Anti-MOG IgG in macaca immune serum was a high-titer Ab.

<p>Macaca MOG HEK were stained with designated titrations of macaca serum (x-axis; 0.1 = 0.1% or 1/1000; 0.0001 = 1/10⁶) collected on days 0, 22, 49, 63, or 93 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110048#pone-0110048-t001" target="_blank">Table 1</a>). A subsequent stain with an allophycocyanin-conjugated anti-macaca IgG secondary reagent was used to detect surface bound anti-MOG Ab.</p

Lewis rats immunized with intra-chain disulfide-linked IgV-MOG developed chronic atypical EAE.

<p>Female Lewis rats aged 8 weeks were immunized with 200 µg of rat IgV-MOG (n = 14) in CFA on day 0. These mice were boosted with 200 µg of rat IgV-MOG in IFA on days 21 and 35. Shown are the individual disease courses for the 12 symptomatic rats (A–L) that exhibited atypical EAE. All clinical data were scored based on atypical scoring criteria. The red arrows (bottom panels) mark the dates of the boosts. Panel M shows the IgV-MOG protein preparation before TCEP reduction (Lanes 1–2, reducing vs native SDS-PAGE, respectively) as well as IgV-MOG after TCEP reduction and refolding (Lanes 3–4, reducing vs native SDS-PAGE). These clinical data are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110048#pone-0110048-t003" target="_blank">Table 3</a>. Histological analyses are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110048#pone-0110048-g004" target="_blank">Figures 4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110048#pone-0110048-g006" target="_blank">6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110048#pone-0110048-t004" target="_blank">Table 4</a>.</p

A CFA/IFA prime-boost strategy with IgV-MOG elicited chronic atypical EAE in Lewis rats.

<p>Lewis rats were immunized with either 50 µg of GP69-88 or 200 µg of rat IgV-MOG in CFA on day 0. Rats were scored daily for clinical signs of EAE. On day 28, GP69-88-immunized rats were or were not boosted with 50 µg of GP69-88 in IFA. Conversely, rat IgV-MOG-immunized rats were or were not boosted with 200 µg of rat IgV-MOG in IFA. Shown are overlapping traces of disease courses of rats immunized with GP69-88 (A), immunized with GP69-88 and given a subsequent boost (B), or immunized with rat IgV-MOG without a subsequent boost (C). Also shown are atypical disease courses of individual rats immunized and boosted with rat IgV-MOG (D–F; 3 of 4 rats shown). Red arrows indicate the booster immunization (B, D–F). The blue line divides the clinical data into classical (left) and atypical (right) courses (D–F).</p

Repeated booster immunizations of IgV-MOG in IFA caused an unusual form of EAE in a cynomolgus macaque.

<p>A cynomolgus macaque was immunized with 500 µg rat IgV-MOG in IFA on days 0 and 22. Booster immunizations that consisted of a mixture of 500 µg rat IgV-MOG and 500 µg macaca IgV-MOG in IFA were given on days 36, 63, and 93. Data represent the full time course of the experiment. After recovery on day 248, the subject did not exhibit additional signs of EAE. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110048#pone-0110048-t001" target="_blank">Table 1</a> provides a timeline for the experimental approach.</p

Summary of CNS histological analyses.

a<p>Histological assessment of classical EAE was assessed in 4 Lewis rats immunized with 50 µg GP69-88 in CFA. Histological assessment of atypical EAE was based on 10 Lewis rats immunized with 200 µg rat IgV-MOG in CFA and boosted with MOG-IgV/IFA on days 21 and 35. Rats were euthanized at peak disease.</p>b<p>Representative sections are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110048#pone-0110048-g004" target="_blank">Figures 4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110048#pone-0110048-g006" target="_blank">6</a>. In rats with classical EAE, approximately 100% of the lesion area in the CNS consisted of punctate areas of mononuclear perivascular infiltration. In rats with atypical EAE, nearly 100% of the lesion area in the CNS was comprised of confluent lesions which were readily discernable by the unaided eye on H&E sections as a dark blue in contrast to the light purple of surrounding normal tissue. Confluent lesions typically involved areas greater than 1 mm² and ranged from 1 mm to greater than 1 cm in one dimension, particularly in the spinal cord or at the base of the brainstem.</p><p>Summary of CNS histological analyses.</p

The immunization and serum collection schedule for the cynomolgus macaque.

a<p>The immunization protocol was comprised of a primary immunization in IFA on day 0 followed by 4 booster immunizations in IFA on the designated days. On each day, subcutaneous injections of rat and/or macaca IgV-MOG were given as 4 separate 100 µl injections (total volume of 400 µl) in the left and right groin and axilla. These immunizations did not result in any visible signs of inflammation at the injection sites. Humane euthanasia was performed on Day 730.</p><p>The immunization and serum collection schedule for the cynomolgus macaque.</p

An intrachain disulfide-linked IgV-MOG protein elicited chronic atypical EAE in rats.

a<p>Lewis rats were immunized with 50 µg GP69-88 (n = 6) or 200 µg rat IgV-MOG (n = 14) in CFA on day 0. On days 21 and 35, rats were boosted with the same respective antigen (50 µg GP69-88 or 200 µg rat IgV-MOG) in IFA. Immunization and boosting with IgV-MOG were performed with an intrachain disulfide-linked IgV-MOG protein. Rats were scored daily for clinical signs of EAE. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110048#pone-0110048-g003" target="_blank">Figure 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110048#pone-0110048-t003" target="_blank">Table 3</a> portray the same experiment. Recovery of rodents was defined as remission to a clinical score of 0 after an episode of paralytic disease.</p><p>An intrachain disulfide-linked IgV-MOG protein elicited chronic atypical EAE in rats.</p