CFS/ME patients exhibiting both the Holmes 
and Fukuda 
diagnostic criteria were selected for this study. The primary endpoint of efficacy, improvement in ET, achieved statistical significance by several analytical methods applied to the ITT cohort. The intra-patient placebo-adjusted enhancement in mean ET at Week 40 was 21.3% (p
0.047). Correction for patients with significant missed doses or dose reductions improved mean intra-patient ET enhancement to 28% (p
0.022). The proportions of patients in the ITT population with changes from mean baseline ET duration at week 40 of at least 25% and of at least 50% were 1.7 and 1.9-fold greater for subjects randomized to rintatolimod than placebo, 39% versus 23% (p
0.016) and 26% versus 14% (p
0.036), respectively. At week 40, the ITT population randomized to rintatolimod had a placebo-adjusted mean intra-treatment group change from baseline ET of 11.8%, 1.8-fold higher than pre-defined in the protocol as the minimum 6.5% required to show efficacy (16–19). Table S8
provides a comparison of the intra-group benefit achieved by rintatolimod to current medical and regulatory standards of care for cardiotropic drugs providing benefit for exertional fatigue symptoms.
There is no specific treatment for CFS/ME and patients utilize a large number of drugs in an attempt to alleviate the debilitating symptoms of the illness. Decreases in concomitant medication usage is an objective measure of the effect of rintatolimod on the symptoms of CFS/ME. Rintatolimod was associated with a statistically significant reduction in the use of concomitant medications used to help treatment symptoms to CFS/ME relative to placebo: 68% of patients receiving rintatolimod decreased use of concomitant medications related to CFS versus 55% of subjects receiving placebo (P
0.048). Other secondary endpoints provided additional evidence of efficacy during Stage 1 and the blinded Stage 2 cross-over. The data from this Phase III trial, moreover, supports an earlier double-blinded, controlled Phase II trial 
, which also demonstrated a greater mean intra-patient ET improvement with rintatolimod compared to placebo (31% vs. 16% (p
0.01)). The decrease in concomitant medication usage associated with rintatolimod treatment has been reported previously to decrease the prolonged cardiac QT intervals induced by many of the drugs used by patients with CFS/ME to alleviate the symptoms of CFS/ME 
Rintatolimod was generally well-tolerated in patients with CFS/ME. The total number of AEs were statistically equivalent between cohorts, although patients receiving rintatolimod had an increased incidence in four AEs (flu-like syndrome, chills, vasodilatation and dyspnea) of usually mild severity. Two deaths, one from each arm of Stage 2 were unrelated to rintatolimod treatment. No significant differences were observed between rintatolimod and placebo in mean hematological, or blood chemistry parameters.
A potential limitation of this trial is that only severely debilitated cases of CFS were enrolled. All patients had a diagnosis of CFS for at least 12 months and a baseline KPS score of 40–60. Therefore, the findings may not be relevant for patients with CFS disease of a lesser severity. A potential source of bias exists since a commercial entity, Hemispherx Biopharma, Inc. funded and conducted this trial using independent investigators. This concern is mitigated since this study remained blinded to all Hemispherx clinical trial staff involved with the study until data base lock and the clinical data and statistical analysis were reviewed and audited by the FDA. This audit included a comparison if the data utilized for the statistical analysis versus the data contained in source documents at the investigator sites.
Differential gene expression in peripheral blood of CFS/ME patients has been reported by a number of investigators using DNA microchip analysis 
. Recently, rigorous patient selection and a microchip that surveys the entire human genome coupled with qPCR gene validation has provided a more complete appreciation of the gene expression profiles that occur in CSF 
. A complex array of differential gene expression can be categorized into functional subsets relating to responses to infection, immunity, inflammation, apoptosis, neurological function, and cancer 
. Many of these differential gene responses are consistent with the large number of infectious agents linked with CFS/ME as well as the altered immune responses and variety of signs and symptoms observed with the disease. For example, eIF4G1 is an eukaryotic mitochondrial translation factor utilized in replication by a variety of viruses including the enteroviruses implicated in the pathogenesis of CFS/ME 
. EIF4G1 variant 5 (GenBank:NM_004953) is upregulated in CFS/ME suggesting a physiological response to viral replication as well as a gene variant favoring pathogen persistence.
Prior to this report, no treatment had been demonstrated to be active in multiple randomized controlled clinical trials. The observed activity of rintatolimod in CFS/ME provides a rational intervention in this complex, multi-factorial disease. Rintatolimod is an activating ligand (dsRNA) for TLR3, which is a first line defense mechanism essential in the induction of innate immunity. Although the mechanism of action of rintatolimod in CFS is unknown, certain genes may be modulated by rintatolimod including those for metabolic enzymes, antiviral response elements, and immune mediators. Subsequent modulation of host defense responses may depend on transient expression of multiple genes and modulation of a number of enzymes regulated by TLR 3 activation. Among the hundreds of upregulated genes triggered by this process 
are the interferons with broad anti-viral activities mediated by interferon's own upregulation of anti-viral genes including 2–5′ adenylate synthetase and protein kinase (p68) activated by rintatolimod 
. The availability of diagnostic assays, based on gene expression profiles, may provide a prognostic genetic signature that will predict response, as well as the opportunity to develop synergistic combinations of rintatolimod and other agents to expand the therapeutic effect in this debilitating disease. The current study more clearly establishes the safety profile of rintatolimod. Other TLRs including those for TLR4 and TLR9 are under development for human use. Their safety, however, based on clinical trials has been questioned based on safety issues, pharmacokinetics, and biodistribution 
. The safety profile of rintatolimod is likely related to the exclusive use of the TRIF intracellular signaling pathway 
as well as the restriction of rintatolimod as a TLR3 agonist, a property not exhibited by the toxic TLR3 ligand, Poly I:C 
. The pathway of TLR3 induction of innate immunity is unique among the TLRs by its exclusive utilization of TRIF as the molecular mediator of NF-κB, IRF-3/IRF-7 transcription factors 
. All the other TLRs utilize a MyD88/TiRAP adapter mediated pathway. The safety profile and unique innate immune stimulating properties of rintatolimod provides strong motivation to assess its potential role in microbial and cancer vaccines. Rintatolimod activates dendritic cells and potentiates both humoral and cell-mediated responses 
. For example, Japanese investigators have demonstrated that rintatolimod has powerful adjuvant activity when coupled with various influenza vaccines including highly pathogenic avian influenza virus (HPAIV) 
. The Japanese government has invested significant effort in providing preclinical data in support of clinical trials of rintatolimod for seasonal and H5N1 HPAIV vaccines.