White Paper EPM Study Updated

Horses with a diagnosis of EPM based on a seropositive result should be evaluated for effectiveness using NeuroQuel® to treat the clinical signs of polyneuritis equi (PNE) when the treatment failed or the horse relapsed after treatment, a condition called post-treatment EPM disease syndrome or PTEDS. The standard of care for EPM diagnosis has shifted away from cerebrospinal fluid (CSF) testing in the past 20 years since the release of Marquis® and Protazil® making reconsideration of the diagnostic criteria of EPM for enrollment into clinical studies necessary.

Pathogenes Inc. is conducting a pilot study to gather data on the effectiveness of NeuroQuel® to control the clinical signs of PNE in horses that have been treated with a licensed anti-protozoal treatment but did not have CSF antibodies confirmed prior to treatment. The study protocol amends the concurred protocol 219-FE-1.1 [012219-G-0044-OT] requiring CSF testing. The amendment is based on the following.

The most recent ACVIM Consensus statement ⁽¹⁾, The Consensus, seeks to provide the veterinary community with up-to-date information of the pathophysiology, diagnosis and treatment of clinically important animal disease. The Consensus identifies “biologic factors such as genetic predispositions of individual animals and parasite-specific factors such as strain differences in virulence remain largely undetermined.”  The highest priority areas The Consensus identified included:

 a) elucidating the immune response in protection and disease;

b) determining how S. neurona causes disease and whether organisms need to be present to cause pathologic changes and clinical signs;

c) elucidating whether S. neurona parasite genotype influences infection and severity of signs;

d) and identifying whether co-infection with other pathogenes can be a contributing factor in EPM cases.

The Committee urged support for the aforementioned projects as the knowledge gained from these studies will lead to earlier and more accurate diagnosis, preventive approaches, and more efficacious treatments.

Justification

The clinical signs of EPM have been suggested as similar to polyneuritis equi (PNE) and requires ruling out EPM as an inclusion criteria for clinical trials evaluating PNE treatments. The cause of polyneuritis equi (PNE) is elusive although the predominant theory is that PNE is an immune mediated inflammatory disease due to a by-stander mechanism ⁽²⁾.  If Sarcocystis sp. elicit immune responses that drive PNE, providing an effective treatment controlling the clinical signs of PNE post-EPM treatment would generate data for one of the highest priority areas identified by the 2016 ACVIM Committee.

Elucidating the immune response in diseased horses

An issue with enrollment into PNE clinical trials post-EPM treatment is the current standard of care for diagnosis of EPM pre-mortem in the United States. Not all veterinarians collect CSF for premortem testing. Pathogenes confirmed with chiefs of large animal clinics at the 2017 EPM session held in Lake Tahoe, NV that veterinary schools no longer teach CSF collection procedures to veterinary students. Most veterinarians in the field do not collect CSF fluid prior to diagnosing EPM preferring to treat and look for a treatment response. An informal survey did not return any veterinarians that routinely obtain CSF taps, those that did work up the horses with CSF taps did not anticipate seeing 6 cases per year due to the rarity of EPM.

There are misunderstandings about the significance of CSF analysis as an indicator of EPM. The Consensus confirms that “CSF is not a definitive indicator of EPM as there is passive transfer across a healthy blood-brain barrier.” casting doubt on the value of CSF analysis. ⁽¹⁾ They further state that horses with higher serum titers are more likely to have detectable antibody levels in CSF.

The preponderance of veterinarians believe there is a diagnostic test for EPM and when a horse is seropositive it should be treated. This view fails to consider the protective role of adaptive immunity and the seroprevalence of Sarcocystis antibodies in the healthy equine population. The choice of EPM-related antigens remain confusing to clinicians. When licensing diagnostic tests USDA prefers a positive/negative status with a positive result being indicative of disease. Most horses in the United States are S. neurona seropositive from Sarcocystis infections initiated in the gut yet they remain clinically normal. No antibody test determines location of the parasite.

Despite the confusion about the relevance of a seropositive status in most horses in the US and the effect of titer on antibody levels in the CSF The Consensus states that “most recent research has focused on the SAG [surface antigen] ELISA’s quantative (end point titers) based on S. neurona surface antigens, these molecules have proven to be good serologic targets in the assays because of their high level of expression in the parasite and their immunogenicity in infected horses.” ⁽³⁾  Danial Howe confirmed that SAG1 of S. neurona is “exceedingly immunogenic, immuno-screening confirmed that 22 of 25 reactive clones contained the  SAG1 gene.” It was shown in three laboratory studies inducing S. neurona SAG1 EPM in horses conducted for Bayer Animal Health (151-330, 151-403, 151-484) from 2001-2003 that antibody development (titer) was statistically correlated with duration of infection and not clinical signs. ⁽⁴⁾ It is recognized that antibodies levels are not an indicator of disease severity in neurological diseases in horses (PNE) and isn’t surprising that S. neurona encephalopathy would be similar that antibody levels don’t correlate with disease severity. ⁽⁵⁾

Authors Stanek et. al. used a SAG5 expressing strain of S. neurona to identify S. neurona in cats residing on horse farms with horses diagnosed with EPM. ⁽⁶⁾ In a collaboration with Dr. David Lindsay at Va Tech, Pathogenes Inc tested 10 seropositive and 10 seronegative feline samples from the Stanek study in a blinded evaluation. Pathogenes Inc determined that the strain of S. neurona used in the assay was a SAG5 expressing strain (SAG1^minus) because all positive samples were seropositive for rSAG5 and the seronegative samples were negative for SAG5. The authors did not detect antibodies against SAG1 or SAG6 in their serum agglutination assay, however The SAG 1, 5, 6 ELISA’s detected antibodies against SAG1 and SAG6 in the samples irrespective of SAG5 status. Dr. Lindsay confirmed that the strain of S. neurona was isolated from a sea otter in 2002 and it was later confirmed as SAG1^minus genotype.

Authors Johnson, Burton, and Sweeney suggest that “the prevalence of SAG1 lacking strains of S. neurona predominate in the mid-Atlantic region accounting for low specificity of SAG1 detection by their tests”. ⁽⁷⁾  Their conclusions were most likely based on omission of SAG1 and SAG6 in their testing. The Consensus confirmed that SAG 1, 5, and 6 are strain-specific to S. neurona and calls into question their position that diagnostic tests based on nonspecific SAG2 and the chimera SAG 4/3 “accurately detect antibodies against Sarcocystis that infect horses”. Until strain specific antigens are used, antibody tests will not be able to serotype the strains of S. neurona that infect horses and therefore won’t be able to answer the question of strain virulence.

Pathogenes Inc determined that most neurologic horses with a gait score of 2 or greater are seropositive to more than one serotype (n=8256) of S. neurona. The predominant single serotype detected in sera was SAG1 (n=218). SAG6 (n=135) and SAG5 (n=90) were identified as a single serotype infections, but less frequently. There was no association with degree of disease and serotype arguing against strain virulence as a factor in EPM horses.

Co-infections that contribute to signs of EPM

A goal of the SAG2, 4/3 ELISA was to dilute out the cross-reactive antibodies against S. fayeri. J S Hoane  determined that S. fayeri is cross-reactive in the SAG 2, 4/3 ELISA saying, “although the calculated (PP) percent positive of a sample values occasionally equaled or eclipsed the cutoffs at one or a few time points, the antibody reactivity was generally below the defined cutoffs for each of the assays.” ⁽⁸⁾ These observations indicate there will be false positive tests due to S. fayeri antibodies on the SAG 2, 4/3 ELISA.

It is generally accepted by veterinarians to discount the impact of S. fayeri on neurological disease in horses. Independently Alleman at UC Davis and Ellison at Pathogenes Inc demonstrated that S. fayeri was a factor in equine neuromuscular disease. ^{(9) (10)}. It is anticipated that S. fayeri displays genotypes that are mutually exclusive as does S. neurona,  therefore Pathogenes Inc uses a disease associated toxin to detect anti-toxin antibodies in horses with clinical signs of disease. Co-infections with both species of Sarcocystis that infect horses were detected by ELISA and S. neurona seropositive sera was more often associate with neuromuscular disease in horses than horses that were positive for S. fayeri. Horses that were seropositive for both Sarcocystis species were more prevalent than single infections and horses with antibodies to both species were significantly more likely to show neuromuscular disease than those with single infections. That is to say horses with co-infections of Sarcocystis were less likely to be sub-clinical but doesn’t indicate if co-infections contribute to EPM. To understand why horses that do not have EPM have clinical signs mistaken for EPM it is necessary to consider the pathogenesis of disease.

Pathogenesis of clinical signs of  EPM

An elevated C-reactive protein (CRP) was detected in the majority of horses with clinical signs of neuromuscular disease (72.6%). ⁽¹⁰⁾ Pathogenes Inc determined in a clinical study that significantly more horses with neuromuscular disease had an elevated CRP when compared to clinically normal horses (p=0.0135) using Fisher’s exact test. It is hypothesized that gut infections with Sarcocystis increase cytokines including IL6 and TNFα. As IL6 increases in infections CRP increases, thus CRP is an indirect indicator of IL6 mediated responses. Immune check-point inhibitors are used in human medicine to block IL6 signaling in a dose dependent reaction.

The direct pathway that allows IL6 to decrease inflammation is through cognate receptors, cells that carry the IL6 receptor are “cognate”. However, IL6 can also be pro-inflammatory. The CRP protein enzymatically releases IL6 receptors from cognate cells, in a dose dependent reaction, and the released soluble IL6 receptor can bind IL6 and induce trans-signaling. ⁽¹¹⁾ IL6 is pro-inflammatory in the presence of elevated CRP via the process of trans-signaling. The pro-inflammatory trans-signaling pathway is initiated by soluble IL6 combining with the CRP-released IL6 receptors. The IL6-IL6 receptor can land on any cell and set up inflammation in opposition to cognate receptors that are limited in their location.

Gut infections with Sarcocystis increases CRP in horses and are closely regulated. The inflammatory response can continue after parasites are eliminated and is posed as the pathogenesis of EPM. It is possible to drive trans-signaling with medications or animal husbandry practices. The initiation of iatrogenic (medical treatment) trans-signaling is evident in horses with high CRP levels. Trans-signaling pathways induced by immune check point inhibitors can lead to steroid resistant autoimmune related side effects that are immune check-point induced disease (ICID). Currently, there is no standardized diagnostic and assessment criteria for ICID and survival rates are low.

Some molecules found on pathogens can induce trans-signaling by their structure. In a collaborative effort with Dr. Stephen Rose-John Pathogenes learned that SAG1 itself cannot induce trans-signaling but an indirect effect of Sarcocystis infections is elevated trans-signaling via CRP. Levamisole HCl was used incriminate IL6-related immunity in sarcocystosis. ⁽¹²⁾ The data based evidence confirms that controlling IL6 is complicated and multi-factorial.

Toxin-secreting S. fayeri strains are associated with elevated CRP levels in horses with clinical signs. ⁽¹⁰⁾ The toxin is not directly detrimental to cells but the toxin induces the production of TNFα that increases CRP thereby inducing the pro-inflammatory trans-signaling pathway. ⁽¹³⁾

Most post-mortem studies conducted in horses with suspected EPM or in EPM challenge studies fail to find the organism, however inflammation is a defining histological finding. ⁽⁷⁾ Based on the difficulty in isolation or identifying Sarcocystis in EPM and the preponderance of inflammatory lesions we assert that the pathogenesis of disease is through innate immune responses specific to IL6 trans-signaling. These observations were supported in the Bayer Studies. ⁽⁴⁾ Dr. Wendel Davis, study coordinator for Bayer, concluded that innate immunity was a factor in disease, SAG1 was a potent stimulator of IFNϒ and based on the study results SAG1 would elicit a protective response against EPM.

Protection against disease

Pathogenes Inc. conducted a study to determine protection afforded by anti-SAG1 antibodies against experimental challenge. Recombinant SAG1 protected horses against SnSAG1 challenge in a laboratory study. ⁽¹⁴⁾ These data support serotype specific protection after infection, horses did not develop EPM. It was evident from the results of several studies that strain virulence in not a factor in developing EPM strain but is directly related to protection. Horses with decreased adaptive immunity due to prolonged anti-protozoal drugs are more at risk to develop EPM.

The relationship between PNE and EPM

The link between Sarcocystosis and PNE is IL6 mediated inflammation. Myelin protein P2 (MP2) displays a T-cell epitope that is the IL6 receptor on MP2. Pathogenes Inc proposes that when chronic inflammation exposes the IL6 receptor to the immune system disease will be apparent. Although ELISA’s that detect anti-MP2 protein antibodies identify a demyelinating neuropathy the assay isn’t expected to correlate with the extent of disease. More investigation is necessary to determine if  the MP2 ELISA will identify horses that will respond to treatments modulate IL6 immunity.

Conclusion

Pathogenes Inc is conducting a pilot study in horses that were originally diagnosed with EPM and were treated with a full course (per label instructions) of and FDA approved treatment for EPM within 90 days of enrollment. Horses that did not receive a CSF analysis prior to EPM treatment will be included in the pilot study and will provide critical data to the EPM community.

References 1-13 available on request.