Identification of CD8 T-cell dysfunction associated with symptoms in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and Long COVID and treatment with a nebulized antioxidant/anti-pathogen agent in a retrospective case series

Abstract

Background

Patients with post-acute sequelae of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection (PASC, i.e., Long COVID) have a symptom complex highly analogous to many features of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), suggesting they may share some aspects of pathogenesis in these similar disorders. ME/CFS is a complex disease affecting numerous organ systems and biological processes and is often preceded by an infection-like episode. It is postulated that the chronic manifestations of illness may result from an altered host response to infection or inability to resolve inflammation, as is being reported in Long COVID. The immunopathogenesis of both disorders is still poorly understood. Here, we show data that suggest Long COVID and ME/CFS may be due to an aberrant response to an immunological trigger-like infection, resulting in a dysregulated immune system with CD8 T-cell dysfunction reminiscent of some aspects of T-cell clonal exhaustion, a phenomenon associated with oxidative stress. As there is an urgent need for diagnostic tools and treatment strategies for these two related disabling disorders, here, in a retrospective case series, we have also identified a potential nebulized antioxidant/anti-pathogen treatment that has evidence of a good safety profile. This nebulized agent is comprised of five ingredients previously reported individually to relieve oxidative stress, attenuate NF-κB signaling, and/or to act directly to inhibit pathogens, including viruses. Administration of this treatment by nebulizer results in rapid access of small doses of well-studied antioxidants and agents with anti-pathogen potential to the lungs; components of this nebulized agent are also likely to be distributed systemically, with potential to enter the central nervous system.

Methods

and Findings: We conducted an analysis of CD8 T-cell function and severity of symptoms by self-report questionnaires in ME/CFS, Long COVID and healthy controls. We developed a CD8 T-cell functional assay, assessing CD8 T-cell dysfunction by intracellular cytokine staining (ICS) in a group of ME/CFS (n = 12) and Long COVID patients (n = 8), comparing to healthy controls (HC) with similar age and sex (n = 10). Magnet-enriched fresh CD8 T-cells in both patient groups had a significantly diminished capacity to produce both cytokines, IFNγ or TNFα, after PMA stimulation when compared to HC. The symptom severity questionnaire showed similar symptom profiles for the two disorders. Fortuitously, through a retrospective case series, we were able to examine the ICS and questionnaire data of 4 ME/CFS and 4 Long COVID patients in conjunction with their treatment (3–15 months). In parallel with the treatment pursued electively by participants in this retrospective case series, there was an increase in CD8 T-cell IFNγ and TNFα production and a decrease in overall self-reported symptom severity score by 54%. No serious treatment-associated side effects or laboratory anomalies were noted in these patients.

Conclusions

Here, in this small study, we present two observations that appear potentially fundamental to the pathogenesis and treatment of Long COVID and ME/CFS. The first is that both disorders appear to be characterized by dysfunctional CD8 T-cells with severe deficiencies in their abilities to produce IFNγ and TNFα. The second is that in a small retrospective Long COVID and ME/CFS case series, this immune dysfunction and patient health improved in parallel with treatment with an immunomodulatory, antioxidant pharmacological treatment with anticipated anti-pathogen activity. This work provides evidence of the potential utility of a biomarker, CD8 T-cell dysfunction, and suggests the potential for benefit from a new nebulized antioxidant/anti-pathogen treatment. These immune biomarker data may help build capacity for improved diagnosis and tracking of treatment outcomes during clinical trials for both Long COVID and ME/CFS while providing clues to new treatment avenues that suggest potential efficacy for both conditions.

Highlights

• •Both Long COVID and ME/CFS patients have dysfunctional CD8 T-cells with severe deficiencies in production of IFNg and TNFa.
• •Immune deficiency and health improve on treatment with nebulized antioxidant, anti-pathogen and immune-modulatory agent.
• •Useful new biomarker, CD8 T-cell dysfunction, assists in diagnosis and tracking response to potential new treatments.

Abbreviations

ALB

albumin

ALP

alkaline phosphatase

ALT

alanine aminotransferase

ANOVA

Analysis of variance

AST

aspartate aminotransferase

BUN

blood urea nitrogen

2B4

Natural killer cell receptor 2B4

CBC

Complete Blood Count

CMP

Comprehensive Metabolic Panel

CTLA4

Cytotoxic T-Lymphocyte Associated Protein 4

CMV

cytomegalovirus

COVID-19

Coronavirus Disease 2019

CRTN

creatinine

EBV

Epstein-Barr Virus

EBNA

Epstein-Barr Nuclear Antigen

FCSB

Flow Cytometry Staining Buffer

FACS

Fluorescence-Activated Cell Sorting

GI

gastrointestinal

HC

healthy control

HC/Unstim

healthy control unstimulated

HC/Stim

healthy control stimulated

HHV6

human herpes virus 6

HSV

herpes simplex virus

IgG

Immunoglobulin G

IgM

Immunoglobulin M

ICS

intracellular cytokine staining

IL

Interleukin

IFN

interferon

LAG3

Lymphocyte-Activation Gene 3

LC/Unstim

Long COVID unstimulated

LC/Stim

Long COVID stimulated

ME/CFS

myalgic encephalomyelitis/chronic fatigue syndrome

ME/Unstim

myalgic encephalomyelitis/chronic fatigue syndrome unstimulated

ME/Stim

myalgic encephalomyelitis/chronic fatigue syndrome stimulated

NF-κB

Nuclear Factor Kappa B

OD

once daily

PBMC

peripheral blood mononuclear cells

PLT

platelet count

PMA

Phorbol-12-myristate-13-acetate

PD1

Programmed cell death protein 1

PC-L1

programmed cell death ligand 1

PEM

post-exertional malaise

POTS

postural orthostatic tachycardia syndrome

RNS

reactive nitrogen species

ROS

reactive oxygen species

RBC

red blood cell

RT

room temperature

SARS-CoV-2

Severe Acute Respiratory Syndrome Coronavirus 2

SOB

shortness of breath

STD

standard deviation

SEM

standard error of the mean

TCR

T-cell receptor

TENS

transcutaneous electrical nerve stimulation

TID

three times per day

TGF-β

Transforming Growth Factor beta

TNFα

tumor necrosis factor alpha

Tx

treatment

ULN

upper limit normal

UMass Chan

University of Massachusetts Chan Medical School

VCA

Viral Capsid Antigen

VZV

varicella zoster virus

WBC

white blood cell

1. Introduction

Severe and long-lasting immunopathologic nervous system and systemic sequelae of acute viral infections have been known to occur for decades (Choutka et al., 2022). Long COVID and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), two highly similar disorders, have recently come to the forefront of medicine following the recent acute coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)(Davis et al., 2023). There are reports that 20–30% of people who had acute COVID-19 infection develop a prolonged syndrome now known as Long COVID (Post-acute sequelae of SARS-CoV-2 infection (PASC)), wherein patients note severe fatigue interfering with daily living and ability to work (95%), post-exertional malaise (PEM) (90%), and many other symptoms associated with ME/CFS, including postural orthostatic tachycardia syndrome (POTS), cognitive difficulties (often referred to as “brain fog”), and unrefreshing sleep (Davis et al., 2021; Hill et al., 2022; Al-Aly et al., 2021; Taquet et al., 2021). This condition appears to begin either as a continuation of the acute COVID-19 episode, or starts a month or more later, often in individuals with mild or moderately severe acute illness who were never hospitalized. Long COVID predominantly affects people between the ages of 20–60 years (Davis et al., 2021; Al-Aly et al., 2021, Hill et al., 2022, Taquet et al., 2021). Half of the individuals with mild or moderate acute COVID-19 symptoms who were still ill after 6 months were recently reported to meet the criteria for ME/CFS (Kedor et al., 2022). Considering that acute SARS-CoV-2 infection causes major dysregulation in the immune system (Huang IP, 2020) it would not be too surprising that the immune system may have difficulty in re-establishing homeostasis in some individuals (Phetsouphanh et al., 2022).

If some Long COVID develops into ME/CFS, it is predicted that there will be an increase of up to 22 million cases of ME/CFS. At this time, it is unknown whether those with Long COVID will ever fully recover from this disorder, or what will be the long-term health consequences including risk of increases mortality. What is without doubt is that this new secondary pandemic will be devastating to the medical system and economy without intervention.

ME/CFS is a debilitating disease with the prevalence in the United States estimated between 519 and 1038 per 100,000 people impacting between 1.7 and 3.7 million people (based on 2017 population data) and as many as 34 million worldwide (Valdez et al., 2019) At least 75% of those diagnosed with ME/CFS are unable to work, 25% report being housebound and 10% are bedbound (Unger et al., 2017; Pendergrast et al., 2016). Currently, an estimated 80% of ME/CFS cases remain undiagnosed (Bested and Marshall, 2015).

A single specific, causative pathogen in ME/CFS has not been found, although certain infectious agents like Epstein-Barr and other herpes viruses, in addition to enteroviruses, have more frequently been considered as potential triggers of ME/CFS (Beyond Myalgic Encephalomyelitis, 2015). Multiple molecular processes appear to be perturbed, especially immunological and metabolic pathways, but the causes of these perturbations, and the reason they are variable between patients and within patients across time, remains unknown (Hornig et al., 2015; Fluge et al., 2016; Armstrong et al., 2014; Naviaux et al., 2016a). Inflammatory processes are implicated in many studies, with evidence of altered inflammatory cytokines in ME/CFS patient serum and plasma; although this indicates significant immune dysregulation, it does not identify the immunopathogenic mechanism and serum cytokines’ high degree of lability limits their utility as biomarkers (Hornig et al., 2015; Broderick et al., 2010; Russell et al., 2016; Fletcher et al., 2009; Hardcastle et al., 2015; Maes et al., 2012a, 2012b). It has been suggested that there is a general pattern of increased inflammation early in the disease and a more immunosuppressed state after long-term illness (Hornig et al., 2015; Russell et al., 2016).

Dysfunctional T cells have been reported in ME/CFS and Long COVID patients as well as during infection with persistent virus such as EBV, HIV and HCV in humans and LCMV in mice (Blank et al., 2019; McLane et al., 2019). During persistent viral infections and cancers in humans and in animal models this CD8 T-cell dysfunction has been well studied and described as CD8 T-cell clonal exhaustion (Blank et al., 2019; McLane et al., 2019). Clonally exhausted CD8 T-cells are characterized by a progressive loss of effector functions, high and sustained inhibitory receptor expression, metabolic dysregulation, poor memory recall and homeostatic self-renewal and distinct transcriptional programs (McLane et al., 2019). The nature of the CD8 T-cell dysfunction in ME/CFS and Long COVID has not been well characterized.

Many ME/CFS patients have had this disease for decades, while Long COVID patients currently have a maximum duration of illness of 3.5 years, given the timing of the SARS-CoV-2 pandemic. Effective therapies for Long COVID patients are critically needed now to help prevent this disorder from becoming a decades-long chronic disease, as with ME/CFS. Inspiritol, a nebulized antioxidant/anti-pathogen agent, was developed, in part, as a broad spectrum, multi-mechanism biopharmaceutical. It is administered as a nebulized liquid therapeutic to address derangements of reactive oxygen species (ROS), aberrant production of type I and II IFNs, and high levels of pro-inflammatory cytokines, key factors involved in the pathogenesis of pulmonary inflammation and cell death that accompany acute respiratory distress syndrome (Maes et al., 2011, 2012a, 2012b, 2021; Dennis et al., 2021; Kumar et al., 2020; Wu, 2020; Al-Hakeim et al., 2023; Fukuda et al., 1994; Paul et al., 2021; Takemoto et al., 2021; Gould et al., 2010, 2011, 2015; Prousky, 2007; Rusznak et al., 2000; Buhl et al., 1990; Dekhuijzen, 2004; Straface et al., 2000; Bridgeman et al., 1991; Hagiwara et al., 2000; Paulin et al., 2017; Lima et al., 2013; Juergens et al., 2003, 2004, 2018, 2020; Rhoden et al., 2004; Kennedy-Feitosa et al., 2016; Dahham et al., 2015; Machado et al., 2018; Kim et al., 2015). These oxidative stress and immune anomalies are well-documented in patients with chronic obstructive pulmonary disease (COPD), asthma, various respiratory viral diseases, ME/CFS, and more recently, in both hospitalized and non-hospitalized COVID-19 and Long COVID patients (). Oxidative stress, cytokine and free radical storms, and T-cell abnormalities, including T-cell dysfunction, are associated with immune system dysregulation, resulting in systemic impacts that include the respiratory, neurocognitive, gastrointestinal, heart, lung, liver, kidney and pancreatic sequelae documented in Long COVID patients (Huang IP, 2020, Phetsouphanh et al., 2022). This nebulized treatment was initially designed for use in patients with COPD, asthma and, more recently, acute COVID, Long COVID and ME/CFS.

The pathogenesis of illness in some subjects with Long COVID may overlap with at least a subset of ME/CFS. A common theme in Long COVID and ME/CFS may be an aberrant response to an immunological trigger, like infection, resulting in a sustained dysregulated immune system because of overactivation of CD8 T-cells and subsequent dysfunction, reminiscent of CD8 T-cell clonal exhaustion. Compounds in the nebulized antioxidant/anti-pathogen agent can act to attenuate Nuclear Factor Kappa B (NF-κB) signaling (Kim et al., 2015; Greiner et al., 2013; Francomano FC et al., 2019; Oka et al., 2000; Sudhoff et al., 2015; Yadav and Chandra, 2017; Cantin et al., 2000; Lou and Kaplowitz, 2007; Xu et al., 2016), potentially decreasing CD8 T-cell abnormalities, including T-cell dysfunction. Clonal T-cell exhaustion can lead to further oxidative stress, and compounds in this nebulized treatment have been shown to decrease oxidative stress by scavenging oxygen and other free radical species (Maes et al., 2011, 2012a, 2012b, 2021; Dennis et al., 2021; Kumar et al., 2020; Wu, 2020; Al-Hakeim et al., 2023; Fukuda et al., 1994; Paul et al., 2021; Takemoto et al., 2021; Gould et al., 2010, 2011, 2015; Prousky, 2007; Rusznak et al., 2000; Buhl et al., 1990; Dekhuijzen, 2004; Straface et al., 2000; Bridgeman et al., 1991; Hagiwara et al., 2000; Paulin et al., 2017; Lima et al., 2013; Juergens et al., 2003, 2004, 2018, 2020; Rhoden et al., 2004; Kennedy-Feitosa et al., 2016; Dahham et al., 2015; Machado et al., 2018; Kim et al., 2015). Both oxidative stress and T-cell dysfunction can lead to poor control of latent and persistent viruses such as herpes viruses, which may all contribute to autonomic instability and neurological abnormalities. Our hypothesis is that immunomodulatory properties in this nebulized antioxidant/anti-pathogen agent may break the cycle of immune system dysfunction and associated oxidative stress responses in ME/CFS and Long COVID by working simultaneously through these multiple mechanisms.

To partially test this hypothesis and to potentially develop a tool for tracking response to therapy, we developed a CD8 T-cell functional assay, specifically assessing CD8 T-cell dysfunction by ICS in a group of ME/CFS and Long COVID patients and comparing them to healthy controls with similar age and sex. To help in future studies designed to track therapy response we also developed a self-report symptom severity questionnaire that further confirmed these two disorders had similar symptom profiles. Then, in a case series of 8 patients treated with the nebulized antioxidant/anti-pathogen agent, we retrospectively reviewed ICS and questionnaire data in 4 ME/CFS and 4 Long COVID patients for evidence of changes in function of their T-cell immune response and symptoms. First, we present a detailed retrospective description during treatment for one individual from each group to highlight the characteristics and severity of these poorly understood illnesses, and to illustrate changes in individuals in parallel with therapy over 15 months. Then, we present retrospective data from the two diagnostic subsets from the biomarker study (ME/CFS, LC) that show significant increases in CD8 T-cell IFNγ and TNFα production and decreased patient-reported symptom severity ratings, without side effects, over 3–15 months of treatment.

2. Materials and methods

Human subjects: All participants were recruited from University of Massachusetts Chan Medical School (UMass Chan) community, UMass Memorial Health Center and from The Salerno Center for Complementary Medicine, New York, New York. Written informed consent was obtained from all participants. ME/CFS patients in this study had previously been diagnosed by Institute of Medicine criteria by their physicians (Beyond Myalgic Encephalomyelitis, 2015). As there are no standardized criteria for Long COVID, we applied a modified CDC definition of PASC (Hill et al., 2022), diagnosing Long COVID patients based on a history of more than 2 months of protracted fatigue unrelieved by rest since having acute COVID-19, and of persistent major symptoms such as brain fog, poor sleep and post-exertional malaise. At each blood testing visit, questionnaires (Supplemental Fig. S9 or S10) were completed by all study participants. Subjects were tracked over time if possible, returning every 1–6 months for symptom review and based on their availability. In order to not be so tiring for these very ill patients and to facilitate the patient and research experience, the UMass Chan Symptom Severity Questionnaire (Suppl Fig. S9&S10) was a more simplified version of the dominant symptoms identified in the more extensive questionnaires frequently used in the assessment of patients for ME/CFS (Unger et al., 2017). Symptom presence, and severity when present, was scored from 0 to 10, with 10 being the most severe; this widened range of scoring options appeared to increase patients’ ability to document gradual changes in severity. The research presented here was approved by the Institutional Review Board committee at University of Massachusetts Chan Medical School, Worcester, Massachusetts.

A subset of 8 patients that had enrolled in the UMCMS study elected separately to pursue treatment with the nebulized antioxidant/anti-pathogen agent under the auspices of a clinical center for complementary medicine. Each of these patients later provided consent to be included in a published retrospective case series. Each patient either had in-person or telemedicine medical consultations prior to being prescribed treatment. Each patient also signed a Medical Information Release Form (HIPAA Release Form) and provided a signed comprehensive medical history, previous medical records, and testing results. Each patient completed the DePaul Symptom Questionnaire-2 (DSQ-2) questionnaire to assess the presence of symptoms, and their severity, and to evaluate the case definition fulfillment of individuals with ME/CFS; the DSQ-2 was also used in this study for Long COVID patients (Bedree et al., 2019). In this retrospective case series we only included those patients who were already enrolled in the UMass Chan Medical School T-cell biomarker study who had separately elected to pursue, and who had stayed on, treatment for 2 months or more and who had elected to return for follow-up questionnaires and repeat blood work to enable a retrospective review of changes in their T-cell biomarkers and in their clinical parameters over time in parallel with their use of the nebulized agent.

Study population: For the UMass Chan Symptom Severity Questionnaire studies we enrolled 20 ME/CFS (16 females, 4 males; mean age: 53.8 ± 3.1 (range 23–77); mean (±SEM) duration of illness: 23 ± 8 years (yr)) and 16 HC (11 females, 5 males; mean age: 55 ± 4 yr (range 22–72)). For the ICS assay used to determine if CD8 T-cell dysfunction was present, we studied a subset of the questionnaire respondents, 10 ME/CFS (7 females, 3 males; mean age: 53.0 ± 3.1 (range 23–72 yr); duration of illness: 22 ± 7 yr) and 10 HC (7 females, 3 males; mean age: 51 ± 3 yr (range 22–72 yr)). The same 8 Long COVID patients were used for the questionnaire and ICS assay studies (6 females, 2 males; mean age: 53 ± 3.3 yr (range 42–64 yr); mean duration of illness: 7.1 ± 1.8 months).

The retrospective case series of 8 nebulized antioxidant/anti-pathogen-treated patients were a subset of the UMass Chan biomarker and symptom profile study. Both the questionnaire and ICS study populations included the 8 treated patients: 4 ME/CFS (all females; mean age: 55.3 ± 6.9 (range 37–69 yr); duration of illness: 24.3 ± 12.9 yr) and 4 Long COVID (all females; mean age: 54.8 ± 4.5 (range 47–64 yr)); duration of illness: 6.0 ± 3.1 months). Table 1 (ME/CFS) and Table 2 (Long COVID) show detailed characteristics of the treated patients including: gender, age, ethnicity, how long they had been ill with ME/CFS or Long COVID prior to treatment, all dates of treatment with the nebulized agent, the number of COVID-19 vaccines a patient had received prior to and including the period of treatment with the nebulized agent, and whether any antiviral medicines were additionally taken in parallel with their elective use of the nebulized agent. Antibody titers for herpes viruses were performed for each patient (Table 1, Table 2). All patients were female and all but one, who is Asian/Pacific, were white. Patients took the nebulized agent for different periods of time, dependent upon the rate of improvement in symptom severity. Long COVID patients were treated for 72–519 days, with 1 of 4 of those patients continuing treatment as of 10/6/2022. ME/CFS patients were treated for 204–519 days and 3 of 4 patients continue to take treatment as of 10/6/2022. Fortuitously, as part of the UMass Chan study, each patient returned for follow-up visits for blood tests and questionnaires every 1–6 months based on their availability while electing to pursue treatment with the nebulized agent. Based on viral blood test results and symptom profiles, LC patient 1, ME patient 1 and ME patient 3 additionally took certain antiviral medicines prescribed by their regular providers during their elective treatment with the nebulized agent, as noted in Table 1, Table 2 Several patients also took doxycycline during the treatment period for either acute Lyme disease or other infections.

Table 1.

ME/CFS patient characteristics in nebulized antioxidant/anti-pathogen study.

Table 2.

Long-COVID patient characteristics in nebulized antioxidant/anti-pathogen study.

Treatment with a nebulized antioxidant/anti-pathogen agent: The nebulized antioxidant/anti-pathogen agent, Inspiritol, is a proprietary nebulized, multi-mechanism medication, initially developed to treat major symptoms of respiratory distress through an agent with combined antioxidant, anti-inflammatory, and broad-spectrum antiviral, antifungal and antibacterial properties. It is a sterile isotonic pH-balanced liquid suspension with four ingredients that are US Pharmacopeia and Drug Master File (DMF)-listed: two endogenously produced compounds (glutathione and methylcobalamin); one repurposed prescription drug that is a pro-drug to an endogenously produced amino acid (N-acetylcysteine); one plant extract monoterpene cyclic ether (1,8-cineole) and one natural bicyclic sesquiterpene (β-caryophyllene), in a proprietary liquid carrier. These five compounds are present in low concentrations (i.e., total of 8.1% (w/w)) in a 90.9% (w/w) saline solution with a DMF-listed emulsifier.

All the active compounds in the nebulized agent are naturally found in foods common in the diet, and each has established values for daily dietary intake. Each of the five active ingredients have individually undergone human clinical trials via the inhalation pathway and have demonstrated efficacy and safety in their use (Prousky, 2007; Buhl et al., 1990; Holroyd et al., 1993; Calabrese et al., 2015; Homma et al., 2012; Bishop et al., 2005; Panahi et al., 2023; Li et al., 2018; Yamada et al., 2023; Williams, 2013; Smith et al., 1952; Kim et al., 2014; Jager et al., 1996). At present, the nebulized antioxidant/anti-pathogen agent is manufactured as a compounded medicine exclusively for a single complementary medicine center (Salerno Center, New York) and is not available through any other source. Inspiritol Inc. has filed documents with the U.S. Food and Drug Administration seeking approval to conduct human clinical trials and has U.S. and foreign patent applications pending. The treatment is inhaled using commercially available nebulizers.

The nebulized agent was prepared as a refrigeration-stable suspension and shipped directly overnight in coolers (to maintain cold temperatures) to patients electing the treatment. The recommended prescribed dose of this nebulized treatment was 5.0 mL TID, taken morning, afternoon and evening. Generally, patients tolerated the treatment well, except for a mild cough in some patients, controlled by decreasing the rate of administration. In retrospective review, patient compliance with this dose was generally good, however, some patients skipped occasional doses or had intermittent use over the course of treatment. Significant deviations from recommended dosing are noted in the Results and Discussion sections.

Clinical laboratory tests for treated patients: In the course of their clinical care, each patient in the retrospective case series had peripheral blood samples tested for complete blood count (CBC) with automated differential; comprehensive metabolic panel (CMP) including liver enzymes and kidney function, thyroid function and herpes family viral serology panels (IgM and IgG for EBV VCA, IgG for EBV EBNA, IgM and IgG for CMV, IgM and IgG for VZV, IgG for HHV6, IgG for HSV1 and HSV2). The above blood tests were conducted at licensed commercial testing companies, including UMass Memorial Health, Accu Reference Medical Laboratory, Quest Diagnostics, Mayo Clinic Department of Laboratory Medicine and Pathology, Lab Corp, Prohealth Laboratory and others. Each patient provided peripheral blood test results for at least two years prior to consultation at the complementary medicine center recruiting for the UMass Chan biomarker study. Each patient included in the retrospective case series had at least one additional peripheral blood test conducted during or after the treatment period.

Peripheral blood mononuclear cell (PBMC) isolation: PBMCs from healthy subjects and patients were isolated from fresh blood samples, processed within 24 h of being drawn, using Ficoll density gradient centrifugation (Ficoll-Paque Plus purchased from GE Healthcare BioSciences, Pittsburgh, PA). The cells were spun at 1800 rpm for 40 min with no brake at room temperature (RT). Most isolated cells were immediately used for CD8 T-cell positive enrichment, and plasma and/or serum samples were subjected to antibody testing. The rest of the cells were cryopreserved in freezing medium +10% DMSO at 10x10⁶ cells/ml at −150^oC in liquid nitrogen.

CD8 T-cell isolation: PBMC from fresh blood, processed within 24 h of being drawn, were counted and re-suspended in anti-CD8 micro-beads (Miltenyi Biotech, Auburn, CA) and MACS buffer, 4 °C phosphate-buffered saline, 2.5 g of bovine serum albumin (Sigma-Aldrich, St. Louis, MO), 2 ml 0.5M EDTA (pH 8.0) (Invitrogen, Grand Island, NY), and then degassed with sterile mesh filter following the manufacturer's protocol as previously described (Aslan et al., 2017). After a 20-min incubation in the dark at 4 °C, PBMCs were washed with MACS buffer and the CD8⁺ T-cells were enriched using Miltenyi Biotech MACS system as previously described (Aslan et al., 2017).

Extracellular staining: Fresh magnet-enriched CD8 T-cells, processed within 24 h of being drawn, were incubated with surface markers for 30 min at RT and subsequently washed twice with flow cytometry staining buffer (FCSB) (500 ml Hank's Balanced Salt Solution with 2% Fetal Calf Serum) and fixed using 100 μl of Cytofix (Becton Dickinson Biosciences, San Jose, CA) for 5 min in the dark at RT. The cells were washed with and re-suspended in FCSB and analyzed by fluorescence-activated cell sorting (FACS) on the LSRII (Becton Dickinson, Waltham, MA). All immunophenotyping surface marker antibodies were purchased from BD Bioscience (CA, USA): CD3 (clone: UCT1), CD4 (clone: SK4), CD8 (clone: SK1). Cells were washed and re-suspended in 300 μl FCSB and analyzed on the LSRII (Becton Dickinson, Waltham, MA).

Intracellular cytokine staining (ICS): The following protocol allows for simultaneous analysis of cell surface and intracellular markers at the single cell level (singlets) by flow cytometry, as previously described (Aslan et al., 2017). To induce production of cytokines IFNγ and TNFα, isolated fresh CD8 T-cells were stimulated for 5 h with PMA/ionomycin in presence of protein transport inhibitors (monensin or brefeldin A solution; BD Biosciences). After staining with cell surface markers (CD3, CD8 and CD4 purchased from BD Biosciences), cells were permeabilized with Cytofix/Cytoperm buffer (BD Biosciences) in accordance with the manufacturer's protocol. Anti-IFNγ (clone B27) and anti-TNFα (clone D-21-1351) antibodies (BD Biosciences) were subsequently used for intracellular staining. Flow cytometry was done with LSRII (Becton Dickinson, Waltham, MA). Control samples to assess for the presence of spontaneous production of cytokines were left unstimulated during the assay and otherwise processed identically in parallel with the PMA-stimulated samples. All ICS data presented in this study are the actual values following stimulation without subtracting spontaneous cytokine production, if any was present.

Statistics: Statistical analyses included multivariate ANOVA with Tukey's multiple comparison test and with corrections for multiple comparisons, paired Student's t-test (two-tailed) and presentation of symptom severity scores and clinical lab test by radar plot.

3. Results

Development of a functional T-cell assay directly ex vivo demonstrating CD8 T-cell dysfunction in both ME/CFS and Long COVID patients. We tested whether CD8 T-cells were dysfunctional in both ME/CFS and Long COVID patients using an assay examining the ability of CD8 T-cells to produce IFNγ or TNFα following stimulation with PMA and ionomycin. We designed a classic ICS assay to provide a direct measure of the functional capabilities of magnet-enriched fresh CD8 T-cells in a format that would be easy to adapt to clinical testing. These functional ICS assays showed that CD8 T-cells of ME/CFS and Long COVID patients had a significantly diminished capacity to produce both cytokines, IFNγ or TNFα, after PMA stimulation when compared to HC as seen in representative FACS plots (Fig. S1) and following statistical analysis of multiple individuals from each group (Fig. 1). HC produced 3.5- or 3.4-fold more IFNγ (HC: 26.1 ± 5.9; Long COVID: 7.4 ± 1.7; ME/CFS: 7.6 ± 2.3) and 3.2- or 4.7-fold more TNFα (HC: 32.2 ± 6.8; Long COVID: 10.2 ± 4.6; ME/CFS: 6.9 ± 2.6) upon stimulation than either the Long COVID or ME/CFS patient group, respectively (Fig. 1). The ME/CFS and Long COVID patients produced a small amount of IFNγ and TNFα, but it was not significantly different from the unstimulated CD8 T-cells. These results are consistent with significant immune dysregulation within CD8 T-cells suggesting that they are highly dysfunctional, although it is not yet clear exactly what persistent antigen, virus, bacteria, fungi or auto-antigen, is keeping these cells in a highly activated state resulting in this dysfunctional state (Cornberg et al., 2013).

Fig. 1.

Dysfunction of CD8 T cells in Long COVID & ME/CFS patients compared to HC. Intracellular cytokine assay (ICS) shows decreased ability of magnet-enriched fresh CD8 T cells of Long COVID & ME/CFS patients to produce IFNγ or TNFα following stimulation with PMA (stim). Individual data points and positive STD are shown. Unstimulated control (unstim). Multivariate ANOVA with adjusted p value for multiple comparisons: **p<0.01, ***p<0.001, ****p<0.0001.

ME/CFS and Long COVID patients have similar symptoms. Based on the UMass Chan Symptom Severity questionnaire ME/CFS and Long COVID patients exhibited similar symptoms and severity scores on their first visit (Fig. 2). Both groups scored at similar severity in fatigue, PEM, sleep problems, headaches, postural hypotension, difficulty concentrating and dysnomia (difficulty retrieving words), the pathognomonic features of ME/CFS diagnosis. Interestingly, self-reported overall scores for the severity of illness were similar for Long COVID (mean: 7.2 ± 0.9; n = 20) and ME/CFS patients (6.7 ± 1, n = 8). Those with Long COVID did report greater severity in muscle and joint pain, chills, and cough. All symptom severity scores for both patient groups were significantly different from HC (p < 0.05) except for vertigo and shortness of breath for both patient groups. Also, ME/CFS patients did not significantly differ from HC in low grade fever and sore throat severity, while Long COVID did not differ from HC in allergy severity (Fig. 2).

Fig. 2.

Severity scoring of symptoms in ME/CFS and Long Covid patients were similar. Patients scored these symptoms on a scale from 0 (no symptom) to 10 (most severe symptom) in questionnaire. ME/CFS n = 20 (blue dots), Long COVID n = 8 (red dots), HC n = 16 (green dots). Individual data points and positive STD are shown. Multivariate ANOVA with adjusted p value for multiple comparisons: ***p < 0.001.

Both the results of the similarity of CD8 T-cell immune dysregulation with T-cell dysfunction and the similarity in symptoms suggested that a similar immunopathologic process may be occurring in these two disorders. Thus, a similar treatment may be beneficial in both. Therefore, we decided to examine in a retrospective case series the ICS and questionnaire data we had obtained over time on the subset of ME/CFS and Long COVID patients that chose to be treated at a complementary medicine center with a nebulized antioxidant/anti-pathogen agent. These patients appeared to be self-reporting significant improvement while on treatment. This treatment, as outlined above, has ingredients that would relieve oxidative stress (Maes et al., 2011, 2012a, 2012b, 2021; Dennis et al., 2021; Kumar et al., 2020; Wu, 2020; Al-Hakeim et al., 2023; Fukuda et al., 1994; Paul et al., 2021; Takemoto et al., 2021; Gould et al., 2010, 2011, 2015; Prousky, 2007; Rusznak et al., 2000; Buhl et al., 1990; Dekhuijzen, 2004; Straface et al., 2000; Bridgeman et al., 1991; Hagiwara et al., 2000; Paulin et al., 2017; Lima et al., 2013; Juergens et al., 2003, 2004, 2018, 2020; Rhoden et al., 2004; Kennedy-Feitosa et al., 2016; Dahham et al., 2015; Machado et al., 2018; Kim et al., 2015), known to be disturbed in ME/CFS and Long COVID; attenuate NF-κB signaling (Kim et al., 2015; Greiner et al., 2013; Francomano FC et al., 2019; Oka et al., 2000; Sudhoff et al., 2015; Yadav and Chandra, 2017; Cantin et al., 2000; Lou and Kaplowitz, 2007; Xu et al., 2016), which would decrease T-cell receptor (TCR) signaling and potentially reverse CD8 T-cell dysfunction; and act directly against pathogens, including viruses, bacteria and fungi (Dahham et al., 2015; Holroyd et al., 1993; Nencioni et al., 2003; Roederer et al., 1990; Geiler et al., 2011; Yang et al., 2010; Li et al., 2016; Astani et al., 2011; Pajaro-Castro et al., 2015; Cavar Zeljkovic et al., 2022; Kim et al., 2022; Jimenez-Guardeno et al., 2022), which we suspect are involved in driving ME/CFS and Long COVID.

To better enhance understanding of these two related complex disorders, we first describe in more detail (Supplemental text) and briefly summarize below the disease and response to therapy for one ME/CFS (ME patient 1) and one Long COVID patient (LC patient 1). These were also the first two patients with these disorders that received this nebulized treatment, who later became part of the retrospective case series; thus, adjustments initially needed to be made in dosage. Perhaps as a result, these two patients have been on the treatment the longest time (more than 15 months). These detailed descriptions of the changes in disease symptoms are compiled from the patients’ self-reported summaries over time.

Case 1: ME/CFS patient improves on a nebulized antioxidant/anti-pathogen agent after relapsing into fourth severe episode of ME/CFS. At presentation, ME/CFS patient, ME patient 1, was a 69-year-old female with ME/CFS diagnosed 47 years previously, with 3 severe episodes of ME/CFS during her lifetime after viral infections (April 1974, March 1984, April 1996). This fourth recent severe episode began following the first dose of the Pfizer-BioNTech COVID-19 vaccine on April 24, 2021. ME patient 1 previously had significant exacerbations often for 2–4 months of her ME/CFS symptoms following vaccines such as tetanus (2009) and influenza (2014), after which she decided not to take any more vaccines. However, she decided because of the severity of acute COVID-19 disease and as a requirement of her employment to take the first dose of vaccine, with devastating consequences. She has no other major medical problems.

ME patient 1 was highly functional working full time as a professional by following a very strict protocol of supplements and pacing. Her self-reported overall severity score for her ME/CFS symptoms prior to vaccination was 2 (Fig. 3A). Similar to our data in ME/CFS patients, her immunological profile showed CD8 T-cell dysfunction as seen in the representative FACS plot (Fig. S1B) and in the mean of 3 different time points taken over 3 years with only 2.3 ± 0.7% (n = 3) of her CD8 T-cells producing IFNγ and 0.01 ± 0.01% (n = 3) producing TNFα upon PMA stimulation compared to 34 ± 10.9% and 25.5 ± 2.8%, respectively, the mean observed of three different time points over 2 years in a healthy individual (HC 1) (Fig. 4, Fig. S1A representative FACS plot). This >90% reduction in cytokine production illustrates the profound dysfunction of her CD8 T-cells.

Fig. 3.

Symptoms improve over time in ME/CFS patient (ME 1) during nebulized antioxidant/anti-pathogen therapy. A. Radar plot shows the change in self-reported score of each symptom at various times over the therapy including overall symptom severity. B. Line plot shows how the most common symptoms of ME/CFS improve over time on therapy and indicate when antiviral and laser therapy were added to treat chronic shingles (VZV), cold sores (HSV-1) and EBV reactivation. Nebulized antioxidant/anti-pathogen treatment (Tx) period5/3/2021 to present. Patient scored these symptoms on a scale from 0 (no symptoms) to 10 (most severe symptoms). First Pfizer-BioNTech vaccine 4/24/2021. Valacyclovir −1/24/2022 to the present. Spironolactone (SPLT) started 3/21/22 to the present. PEM-post exertional malaise, GIgastrointestinal, dysnomia (difficulty retrieving words).

Fig. 4.

ME/CFS (ME 1) (B) and Long COVID (LC 1) (C) patients show reversal of CD8 T cell dysfunction as evidenced by significantly improved IFNγ and TNFα production following PMA stimulation (stim) in ICS assay. Data shows the mean of ICS assays which were done sequentially 2–3 times before nebulized antioxidant/antipathogen treatment and 5–6 times post-treatment (Tx) over a 15-month period (between 5/26/21-7/1/22). Mean of 3 different time points over 2 years of healthy control (HC 1) (A) shows that ICS findings are consistent in HC. Unstimulated control (unstim). Pre = Pre-treatment, post = post-treatment. Individual data points and positive STD are shown. Multivariate ANOVA with adjusted p values for multiple comparisons *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Within 48 h after the SARS-CoV-2 vaccine her left arm was extremely sore and swollen (where she received the vaccine), with biceps muscle fasciculations, and left axillary adenopathy. Within one week she had massive total body extremely tender lymphadenopathy (10+), muscle and joint pain, increased bruising and bilateral conjunctivitis, a splitting headache worsened by positioning her head downwards, extreme fatigue and PEM, sleeping most of the time, and narcolepsy following eating. She progressively worsened such that by day 10 post vaccine (5/4/21) she felt like she was in a cytokine storm, barely able to speak because of brain fog and word retrieval problems, wobbly with a wide-based gait, as she was not certain of her footing and her head began bobbing much like a patient with Parkinson's Disease. At this time, the patient self-reported an overall severity score of 10/10. She also scored PEM, lymph nodes, sleep problems, headaches, difficulty concentrating and dysnomia (difficulty retrieving words) at 10/10 in severity (Fig. 3A and B). Instead of calling an ambulance she start a nebulized antioxidant/anti-pathogen agent. Within 1½ hours of her first nebulizer treatment most of her very severe symptoms decreased (headache, brain fog, difficulty walking, head bobbing and passing out after eating), and she felt some increase in energy. This improvement was such that she did not need hospitalization and she was able to function well enough that she could just care for herself while living alone (Fig. 3).

In order to receive an exemption at her workplace from a second dose of COVID vaccine she was required to see a neurologist. The neurologist diagnosed her with a neuro-inflammatory condition based on her symptoms and findings. The major clinical finding at this time was evidence of complete inability to feel the vibration of a tuning fork in both feet (impaired vibration sense) and her initial acute onset post vaccine of inability to tell where she was placing her feet, consistent with possible either central or peripheral neuropathology and involvement of the proprioception pathway. (At two years post vaccine while still on the nebulizing therapy the patient had return of her ability to feel the tuning fork in both feet and improved ability to tell the position of her feet more like her normal prior to the vaccine). The magnetic resonance imaging studies showed multiple neuro-inflammatory lesions bilaterally in the subcortical regions of her brain. At this time the patient's cognitive function was still impaired as she could not play Sudoku at all, nor could she write papers and these abilities did not return until 7 months on therapy following the vaccine.

She was gradually improving on the nebulized therapy when in November 10–24th, 2021 (week 31–32) the treatment was stopped for 2 weeks due to issues with getting ingredients at the compounding pharmacy. During this time the patient greatly regressed with increased fatigue, and headaches, unable to work. During this interruption in therapy she also developed symptoms of chronic shingles on the right side of her back, presenting with burning and tingling feeling with very mild redness and induration (no vesicles) (“zoster sine herpete”), which is more common in immune-compromised individuals, and chronic cold sores on the right side of her upper lip. She had elevated levels of VZV IgG antibodies, HSV1 antibodies, and EBV VCA IgG and EBNA IgG, consistent with reactivation of these viruses as well as her clinical diagnosis of chronic shingles and cold sores (Table 1). She restarted the nebulized treatment and started valacyclovir 1000 mg three times a day for 2 weeks and then 1000 mg once daily (OD). She also started spironolactone on March 3rd, 2022 based on the findings of Swaminathan and colleagues (Verma et al., 2020) that this mild diuretic could also inhibit EBV lytic protein transcription and thus prevent reactivation of the virus.

The patient continued to gradually improve on this triple therapy of nebulized antioxidant/anti-pathogen agent, valacyclovir and spironolactone; after 15 months she was close to her pre-vaccine level of symptom severity. Fig. 3A summarizes in a radar plot the gradual decrease in severity of her main symptoms, such as fatigue, PEM, muscle and joint pain, lymph node enlargement and tenderness, sleep quality, headaches, postural hypotension, difficulty concentrating, dysnomia, and allergy-type symptoms in 8 visits over 15 months. Her overall severity score of 10 prior to treatment returned to her baseline of 2 (Fig. 3A). Fig. 3B in a linear scatter plot shows how the severity of her more neurological-type symptoms, including fatigue, headache, difficulty concentrating, sleep problems, PEM, postural hypotension and dysnomia, had decreased to scores of 2–5 at 5 months after therapy, from initial scores of 8–10 prior to treatment. By 15 months of triple therapy these symptom scores were at a level of 0–2. Her chronic shingles and cold sores still continue to slowly resolve.

Throughout her symptom improvement she had a consistent improvement in the CD8 T-cell dysfunction assay employed here (Fig. 4, Fig. S1B). The representative FACS plot shows that her CD8 T-cell IFNγ production upon PMA stimulation prior to treatment was 2.9% and increased to 11.1% post-treatment, while her TNFα went from 0.06% to 16.1%. She had a sustained 5-fold increase in her production from 2.3 ± 0.7% (mean of 3 time points over 3 years prior to treatment) pre-treatment to 10.7 ± 2.0% (mean of 6 different time points over 15 months) post treatment, and her TNFα production had gone from essentially zero pre-treatment to 10.3 ± 2.3% post-treatment (mean of 6 time points over 15 months) (Fig. 4). These results suggest the patient may have been able to recover from a severe episode of ME/CFS brought on by the COVID vaccine. In fact, her pre-vaccine CD8 T-cell dysfunction, which may have contributed to her severe reaction to the vaccine, has at least partially reversed while she continues treatment.

Serological studies showed that following the first COVID vaccine, ME/CFS patient ME patient 1 did develop antibodies by ELISA to the SARS-CoV-2 spike protein but had no evidence of neutralizing antibody. This patient has no history of having acute COVID-19, her viral tests by PCR have always been negative and she had no SARS-CoV-2 nucleocapsid antibodies by ELISA, which only the virus infection can induce (not the current mRNA vaccines). Thus, this ME/CFS patient shows evidence that the potential immunological biomarkers that we have identified in this disease may parallel symptomatic improvement during treatment and appear to be moving in the direction of more normal responses as the patient improves. This patient had no side effects from the treatment and her routine blood work (CBC with complete differential, liver and kidney function, thyroid function) remained normal following 15 months of continuous treatment with the nebulized agent described here (Fig. 5).

Fig. 5.

Blood chemistry and CBC of ME/CFS patient (ME 1) remains normal throughout nebulized antioxidant/anti-pathogen treatment (Tx) as shown on a radar plot. WBC- white blood cell, RBC –Red Blood Cell, PLT – Platelet Count, BUN – Blood Urea Nitrogen, CRTN – Creatinine, ALB – Albumin, ALP – Alkaline Phosphatase, ALT – Alanine Aminotransferase, AST - Aspartate Aminotransferase, ULN- upper limit of normal.

Case 2: Long COVID patient (LC patient 1) improves on a nebulized antioxidant/anti-pathogen agent. This case involves a 61-year-old female who was healthy until she suffered symptomatic acute COVID-19 infection on November 23, 2020, with severe fatigue, chills, cough, headache, laryngitis, loss of smell and extreme muscle and joint pain. She recovered at home over 2 weeks, but within 4 weeks after acute COVID she developed symptoms of Long COVID with ongoing severe fatigue, brain fog, post exertional malaise, headache, laryngitis, feeling freezing cold, and extreme muscle and joint pain, increased bruising and difficulty walking and suddenly passing out and sleeping for hours after eating, like the clinical phenomena described for ME patient 1. She was a fully self-employed professional who was unable to work and support herself after the acute COVID-19 infection. In 3/3/21 her severity scores for overall symptom severity and for the individual symptoms of dysnomia, low grade fever, cough, and sore throat were 8/10; her severity scores for difficulty concentrating, chills, lymph nodes, gastrointestinal symptoms, muscle and joint pain were 10/10; and her headaches, PEM, postural hypotension and sleep difficulty symptom severity scores were between 3 and 5.

She then had a severe immune overactivation following the first dose of the Pfizer-BioNTech COVID-19 vaccine on April 23, 2021, further aggravating her Long COVID symptoms. This was accompanied by development of marked total body tender lymphadenopathy, very similar to ME patient 1. It is thought that immune overactivation like that described above can lead to T-cell dysfunction as discussed earlier (McLane et al., 2019). Her overall symptom severity score increased from her Long COVID baseline of 8–10. Her headaches, PEM, postural hypotension and sleep difficulty severity scores increased to 6–8 (Fig. 6). At this time, 12 days after receiving the SARS-CoV-2 vaccine, her CD8 T-cells were extremely dysfunctional producing essentially no IFNγ or TNFα upon stimulation (Fig. 4, Fig. 1C representative FACS plot).

Fig. 6.

Symptoms improve over time in Long COVID patient (LC 1) during nebulized antioxidant/anti-pathogen therapy with exacerbation during second COVID-19 vaccine. A. Radar plot shows the change in self reported score of each symptom at various times over therapy. B. Line plot shows how the most common symptoms of ME/CFS and Long COVID improve over time on therapy and indicate when vaccines were given, and antiviral therapies to treat reactivated herpes viruses VZV, EBV and CMV. Nebulized treatment (Tx) period-5/5/2021 to present with 2 wk break 11/10/21-11/24/21. Patient had acute COVID-19 on 11.23.20 and there after developed Long COVID. Patient scored these symptoms on a scale from 0 (no symptoms) to 10 (most severe symptoms). First PfizerBioNTech vaccine was given 4/23/2021 and second BioNTech-Pfizer COVID-19 vaccine on 1/21/22. Valacyclovir - for 2 weeks (1/26/2022 to 2/9/2022). Spironolactone (SPLT) started 3/21/22 to the present. valganciclovir 3/21/2022 to 7/1/2022. PEM-post exertional malaise, GI-gastrointestinal.

Twelve days (5/5/21) after the vaccine she started the nebulized therapy. Within 1½ hours of the first dose, she had a dramatic decrease in many of her severe symptoms including brain fog, headache, wobbly walk and her laryngitis. Most of her other symptoms gradually improved over the next few weeks, including passing out after eating, and the severe cold feeling. On May 26, 2021 (Day 21 on treatment) the patient reported that her joint and muscle pain severity had decreased from 10 to 5; muscle tightness had decreased from 10 to 5; brain fog decreased from 9 to 4, and headaches decreased from intermittent daily to infrequent and mild/manageable (Fig. 6). She was able to resume some work, such as client meetings within two weeks of starting the nebulized treatment.

This patient has continued to use the nebulized antioxidant/anti-pathogen agent through to the present time and has reported her symptoms have continued to improve.

However, in November (7 months after beginning treatment), when the nebulized agent was not available for 2 weeks, the patient had a significant exacerbation of all her symptoms like ME patient 1. In fact, she had difficulty when re-starting the nebulized agent, as it did not give her the same level of increase in energy that she had previously. She had more problems tolerating the treatment, particularly with cough and a difficult to describe unpleasant feeling. She continued treatment as it did increase her energy level. She was found to be IgM + for EBV VCA and CMV; HHV6 and VZV IgG titers were also elevated (Table 2). This would suggest that treatment with the nebulized agent may have been helping to keep these viruses under some control whereas temporary discontinuation of treatment for 2 weeks may have allowed these latent viruses to profoundly reactivate.

Prior to being treated with anti-viral agents LC Patient 1 decided to take her second dose of the Pfizer-BioNTech COVID-19 vaccine. The vaccine caused all her symptoms of Long COVID to exacerbate although not as severely as before (Fig. 6). Her symptoms also improved much more rapidly than after the first vaccine as she had continued the nebulized treatment at the time the second vaccine dose was administered. She was treated with valganciclovir 900 mg daily for 15 weeks for her apparent CMV and HHV6 reactivation, along with spironolactone 12.5 mg daily for EBV reactivation. Within a week of starting the valganciclovir she found that the nebulized agent began working as well as it had before and she subsequently returned to work almost full time. Serological studies showed that following the first COVID vaccine, LC patient 1 did develop good neutralizing antibody responses to SARS-CoV-2.

Like ME patient 1, during treatment with the nebulized agent, while her symptoms were gradually improving, LC patient 1 had a sustained increase in her CD8 T-cell IFNγ production, going from essentially none pre-treatment to 12.4 ± 2.5% post-treatment (mean of 5 different time points over 15 months), and her TNFα production went from levels below detection pre-treatment to 15 ± 6% post-treatment (mean of 5 different time points over 15 months) (Fig. 4C, Fig. S1 representative FACS plot). Thus, we found evidence over the course of treatment of LC patient 1 that the potential immunological biomarker that we have identified in this disorder, CD8 T-cell dysfunction, may be tracked as it normalizes during treatment becoming more normal as the patient improves. This patient had no side effects from the treatment other than some cough from the nebulizer, which was controlled by nebulizing at a slower rate, and a short spell of an unpleasant feeling when restarting the nebulized therapy following a 2-week break due to production issues, during which she experienced apparent reactivation of multiple herpesviruses. Her routine blood work (CBC with differential, liver and kidney function, thyroid function) following 15 months of continuous nebulized treatment remains normal (Fig. 7). In fact, her liver function studies, which were slightly elevated by the acute COVID infection, were normal after treatment.

Fig. 7.

Blood chemistry and CBC of Long COVID patient (LC 1) remains normal throughout nebulized antioxidant/antipathogen treatment (Tx) as shown on a radar plot. Elevated ALT and ALP on 6/29/2022 from acute COVID-19 infection, subsequently decreased while on inhalation therapy. WBC- white blood cell, RBC –Red Blood Cell, PLT – Platelet Count, BUN – Blood Urea Nitrogen, CRTN – Creatinine, ALB – Albumin, ALP – Alkaline Phosphatase, ALT – Alanine Aminotransferase, AST - Aspartate Aminotransferase, ULN- upper limit of normal.

ME/CFS and Long COVID patients show reversal of CD8 T-cell dysfunction and symptomatic improvement during treatment with a nebulized antioxidant/anti-pathogen agent.

The above retrospective results suggest these two patients (ME patient 1, LC patient 1) were able to largely recover from their disorder, one with chronic ME/CFS that developed into a severe episode following an aberrant immune response to a SARS-CoV-2 mRNA vaccine, and the other recovering from severe Long COVID that was further exacerbated twice after administration of COVID mRNA vaccines. At the same time both patients had evidence that a potential immunological biomarker that we have identified in these disorders, CD8 T-cell dysfunction, decreased in parallel with treatment as the patients symptomatically improved. There is an urgent need for treatment for both disorders; based on these results and the absence of major side effects we retrospectively examined the ICS and questionnaire data of the 6 other patients, 3 ME/CFS and 3 Long COVID patients, who had elected to use the nebulized antioxidant/anti-pathogen therapy and for whom we had sequential symptom severity score and immunological data (Table 1, Table 2). These other 6 patients have been treated for a shorter period than ME patient 1 and LC patient 1, ranging from 3 to 9 months; all were on the higher dose of 5 ml TID. The length of time these patients were symptomatic with LC post-acute COVID-19, prior to treatment is listed in Table 2. All 6 of these other patients showed improvement in symptoms with some variation in which symptoms improved to a greater degree for any individual (detailed radar plots of symptom severity scores of each individual shown in Supplemental Figs. S2–S7). They also had no significant side effects other than mild cough with nebulizer use in some patients, and their metabolic profiles and CBC remained normal (Supplemental Figs. S2–S7).

The data of all 8 patients treated with the nebulized agent (4 ME/CFS and 4 Long COVID; includes ME patient 1 and LC patient 1) showed a mean of 4.4 points (out of 10 points) in self-reported overall symptom severity scores after treatment (54% decrease) (Fig. 8A). In one of the most common symptoms of both disorders, PEM, there was a mean decrease of 3.9 points following treatment (54% decrease) (Fig. 8B). Two self-reported symptoms included dysnomia (Fig. S8B) and difficulty concentrating (Fig. S8B) were also reduced after treatment with the nebulized agent, with a mean decrease of 3.8 points (64% decrease) (Fig. S8A) and mean of 3.9 points (73.8% decrease) following treatment, respectively. Numbers in this retrospective case series were small; however, future work in controlled clinical trials needs to address whether Long COVID patients respond more rapidly to treatment, versus the possibility that some Long COVID patients may improve without treatment (Fig. 9).

Fig. 8.

Significant decreases in self-reported overall symptoms (A) and PEM (B) scores in ME/CFS (red symbols, n = 4) and Long COVID (blue symbols, n = 4) patients following nebulized antioxidant/anti-pathogen treatment. Patients scored these symptoms on a scale from 0 (no symptoms) to 10 (most severe symptoms). Paired t-test (two-tailed) indicates significant differences pre- and post-treatment. Right side of graph shows the difference in score between pre- and post-treatment (Tx) for each patient. PEM-post exertional malaise.

Fig. 9.

Mean value of Long-COVID (A) and ME/CFS (B) patient symptom scores pre and post nebulized antioxidant/anti-pathogen treatment (Tx). Patients scored these symptoms on a scale from 0 (no symptoms) to 10 (most severe symptoms). Long COVID and ME/CFS patients had an overall mean reduction of all symptoms equally weighted of 75% and 52%, respectively. MECFS n = 4, long COVID n = 4.

There also was evidence of improvement in CD8 T-cell dysfunction, with a 3.6-fold increase (p = 0.006, n = 7) in the frequency of CD8 T-cells producing IFNγ by a mean difference of 9.5 ± 2.3.0% going from 3.7 ± 0.9% pre-treatment to 13.2 ± 1.7% post-treatment. We also saw a 2.5-fold increase (p = 0.016, n = 7) in the frequency of CD8 T-cells producing TNFα by a mean 8.8 ± 2.6% going from 5.9 ± 2.3% pre-treatment to 14.8 ± 1.1% post-treatment (Fig. 10). Post-treatment, these values are not yet reaching the mean observed in HC but are approaching levels seen in some individual healthy controls at the lower range of HC, who had a mean of 26.1 ± 6.0% of IFNγ-producing CD8 T-cells and a mean of 32.2 ± 6.8% of TNFα-producing CD8 T-cells (Fig. 1). This retrospective case series suggests that not only was there an improvement in the severity of individual and overall symptoms in both ME/CFS and Long COVID patients; in addition, we noted an improvement in immune dysregulation (decrease in CD8 T-cell dysfunction) that paralleled treatment with the nebulized agent described above.

Fig. 10.

Reversal of CD8 T cell dysfunction evident from increase in INF-γ (A) and TNF-α (B) cytokine production in ICS following PMA stimulation in 4 ME/CFS (red symbols) and 3 Long COVID (blue symbols) patients following nebulized antioxidant/anti-pathogen treatment. Red dots and blue dots show results of ME/CFS and Long COVID patients, respectively. Paired t-test (two-tail) analyses. Right side of graph shows the difference in CD8 T cell % between pre- and post-treatment (Tx) for each patient. Long COVID patient (LC 2) data not available as blood sample was degraded during shipping. (only one selected time point pre- and post-treatment is shown for each patient).

4. Discussion

These retrospective case series results suggest there may be both symptomatic relief and improvement of a type of CD8 T-cell dysfunction, in subjects with two disorders with similar manifestations, ME/CFS and Long COVID, over the course of treatment with a multi-mechanism nebulized agent with antioxidant, anti-pathogen and likely immunomodulatory properties. These findings provide evidence of a potential immunological biomarker, CD8 T-cell dysfunction, for diagnosis and for monitoring of symptoms and treatment outcomes in both ME/CFS and Long COVID. There was no evidence of serious adverse events or laboratory anomalies with this treatment. It is noteworthy that 8/8 patients had a positive response, some within hours of treatment. Intervening in Long COVID patients at earlier stages after the inciting infection potentially holds greater potential to reverse the course of disease, as compared with later stages or with more chronic ME/CFS patients. The therapy may also allow ME/CFS and Long COVID patients to have increased functionality in their daily life, as shown by the setbacks that occurred when some of the patients (ME1 and LC1) temporarily stopped treatment. This suggests that even if many Long COVID patients were to eventually improve without treatment, the nebulized therapy would give them symptomatic relief that would allow them to return to a more normal active life sooner. The preliminary safety, efficacy and biomarker data presented here on the use of this nebulized agent in patients with ME/CFS and Long COVID, in the face of the absence of defined treatments for either disorder, also support moving this novel treatment strategy into appropriately powered, controlled clinical trials to address both disabling conditions.

Most patients in the retrospective case series reported improvement right away in sleep with this nebulized treatment, a factor considered crucial for recovery from ME/CFS. Though data are limited from this small case series, the individual profiles show that there appears to be variability as to which symptoms improve, when, and at what rate. This may relate to the individual immunological compensations each patient has made as their immune systems struggle to control their individual specific set of persistent pathogens, and depending on their overall immune reserves at the time the nebulized treatment was begun. However, we do not know if some patients with more protracted and severe immune dysregulation may require continued treatment, much like individuals with diabetes require insulin to continually correct metabolic defects. Our results, though based on a small number of patients, nonetheless suggest that the earlier we intervene with treatment in Long COVID patients, and in ME/CFS patients with recent onset of disease, the greater their chances of recovery.

A nebulized antioxidant/anti-pathogen agent with potential immunomodulatory properties was used in this small retrospective case series of ME/CFS and Long COVID patients, as it contains multiple components that can act against the multiple mechanisms postulated to underlie these complex disorders. All compounds in this treatment have antioxidant properties, which have been shown to decrease oxidative stress and inflammatory processes, a process previously demonstrated to be a major problem in COVID-19, ME/CFS and Long-COVID (Maes et al., 2011, 2012a, 2012b, 2021; Dennis et al., 2021; Kumar et al., 2020; Wu, 2020; Al-Hakeim et al., 2023; Fukuda et al., 1994; Paul et al., 2021; Takemoto et al., 2021; Gould et al., 2010, 2011, 2015; Prousky, 2007; Rusznak et al., 2000; Buhl et al., 1990; Dekhuijzen, 2004; Straface et al., 2000; Bridgeman et al., 1991; Hagiwara et al., 2000; Paulin et al., 2017; Lima et al., 2013; Juergens et al., 2003, 2004, 2018, 2020; Rhoden et al., 2004; Kennedy-Feitosa et al., 2016; Dahham et al., 2015; Machado et al., 2018; Kim et al., 2015). Although the underlying mechanism for ME/CFS pathogenesis is not known, some research studies suggest that ME/CFS is a disease of chronic inflammation driven by factors such as mitochondrial dysfunction and oxidative/nitrosative stress (Pall, 2003; Morris et al., 2016). Metabolomics studies suggest hypometabolism occurs in ME/CFS, including evidence of downregulated glycolysis (Fluge et al., 2016; Armstrong et al., 2014; Naviaux et al., 2016a, 2016b)). Other studies suggest that disruption of the TCA cycle with impaired pyruvate dehydrogenase (an enzyme in the mitochondrial matrix that catalyzes the TCA cycle (Fluge et al., 2016)) and elevated lactate and low citrate play a role in ME/CFS (Yamano et al., 2016). Previous clinical trials in both ME/CFS (Wood et al., 2021) and Gulf War Illness (Golomb et al., 2014; Baraniuk et al., 2013; Hodgin et al., 2021; Van Doren et al., 2022), a disorder with some shared clinical features, suggest that antioxidants could be beneficial in these conditions.

All the components in this nebulized agent have been reported to have broad direct anti-viral properties against several viruses, including several that routinely produce respiratory symptoms, including parainfluenza-1, influenza A (H5N1), herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2), human immune deficiency virus (HIV-1), and dengue virus 2 (DENV-2) (Holroyd et al., 1993; Nencioni et al., 2003; Roederer et al., 1990; Geiler et al., 2011; Yang et al., 2010; Li et al., 2016; Astani et al., 2011; Pajaro-Castro et al., 2015). 1,8-cineole, β-caryophyllene and methylcobalamin each have antiviral properties to SARS-CoV-2 (Cavar Zeljkovic et al., 2022, Jimenez-Guardeno et al., 2022, Kim et al., 2022). β-caryophyllene has broad spectrum antibacterial properties (in vitro) against several bacterial species, including S. aureus, K. pneumoniae, and P. aeruginosa, and in vivo for M. pneumoniae (Dahham et al., 2015). β-caryophyllene also has broad spectrum antifungal properties against several fungal species (Dahham et al., 2015). As many different persistent pathogens have been associated with ME/CFS as potentially causative or contributing to disease via reactivation, viruses such as EBV, CMV, HHV-6, HHV-7, HHV-8, (Unger et al., 2017; Buchwald et al., 1992; Ablashi et al., 2000), human parvovirus B19 (B19V), enteroviruses (O'Neal and Hanson, 2021; Chia and Chia, 2008), lentivirus, and bacteria (Beyond Myalgic Encephalomyelitis, 2015) such as mycoplasma, lyme and Q-fever, this nebulized agent could potentially help dampen pathogen load of multiple infectious agents at the same time. This nebulized treatment also has multiple ingredients capable of acting as immune modulators through their ability to attenuate NF-κB signaling, a process essential for signaling through the TCR, thus decreasing over-activation and potentially averting subsequent dysfunctional CD8 T-cells (Kim et al., 2015; Greiner et al., 2013; Francomano FC et al., 2019; Oka et al., 2000; Sudhoff et al., 2015; Yadav and Chandra, 2017; Cantin et al., 2000; Lou and Kaplowitz, 2007; Xu et al., 2016; Wenzel et al., 2021; Attiq et al., 2021; Davies et al., 2021).

We propose that an inappropriate immune response to an infection triggers Long COVID and at least a subset of ME/CFS, resulting in chronically dysregulated immune responses with CD8 T-cell dysfunction. For instance, inappropriate immune responses to viruses can occur in the presence of heterologous immunity and activation of cross-reactive CD8 T-cells (Welsh and Selin, 2002; Selin et al., 2011). Cross-reactive activation of memory influenza A (IAV)-specific memory CD8 T-cells in HLA-A2+ patients during acute EBV infections result in the highly dysregulated immune response and syndrome of acute infectious mononucleosis, wherein there is massive over-expansion of functionally exhausted CD8 T-cells in conjunction with high viral load persisting for life (Aslan et al., 2017; Clute et al., 2005). A highly dysregulated immune response may lead to viral persistence, CD8 T-cell dysfunction and/or T-cell clonal exhaustion.

Clonally exhausted CD8 T-cells are characterized by a progressive loss of effector functions, high and sustained inhibitory receptor expression, metabolic dysregulation, poor memory recall and homeostatic self-renewal and distinct transcriptional programs (McLane et al., 2019). For CD8 T-cell clonal exhaustion to occur there needs to be persistent antigen from virus or tumor that drives hyperactivation of T-cells, leading to sustained co-expression of multiple inhibitory receptors on T-cells e.g., programmed cell death protein 1 (PD1), cytotoxic T-lymphocyte associated protein 4 (CTLA4), natural killer cell receptor 2B4 (2B4), lymphocyte-activation gene 3 (Lag 3). These inhibitory receptors provide negative costimulatory signals to CD8 T-cells that prevent optimal CD8 T-cell effector responses. However, responses to persistent antigen, virally infected cells, immunoregulatory cells, antigen presenting cells, and tumors can contribute to a chronic state of inflammation by producing both inflammatory cytokines, like interferon alpha/beta (IFN $\alpha$/β) and inhibitory cytokines, like interleukin-10 (IL-10) or transforming growth factor beta (TGFβ), further driving exhaustion by eliciting negative regulatory signals on T-cells, either directly or indirectly. The ability to reinvigorate exhausted CD8 T-cells through inhibitory receptor blockade (check-point inhibitors) such as anti-PD1, anti-programmed cell death ligand 1 (PDL1) and anti-CTLA4 highlights the therapeutic potential of better understanding dysfunctional T-cells (Barber et al., 2006; Pauken et al., 2019; Mellman et al., 2016). Thus, we will focus in future work on extending current findings of T-cell dysfunction to determine whether other features associated with T-cell clonal exhaustion are also present in ME/CFS and Long COVID, through investigation of specific markers such as PD1 and RNA seq expression patterns. We used a general trigger, PMA, in the well-established ICS assay system in studies here to examine over all functionality of the CD8 T-cells, which has the advantage of not being antigen or HLA-restricted making it easier to utilize as a clinical biomarker assay. Studies interrogating T-cell responses to specific pathogenic triggers (SARS-CoV-2, EBV, others) are also likely to be informative about the disease process.

Similar to our results on CD8 T-cell dysfunction, a recent report based on 215 patients, using extensive state-of-the-art single cell RNA-sequencing immune profiling, found evidence consistent with CD8 T-cell clonal exhaustion and evidence of herpes virus reactivation to be two of the hallmark findings in the immunopathogenesis of Long COVID (Klein et al., 2022). A recent study from Hanson and colleagues that examined T-cell metabolism and cytokine associations found that ME/CFS CD8 T-cells had reduced mitochondrial membrane potential compared to healthy controls (Mandarano et al., 2020). CD8 T-cells from ME/CFS patients had reduced glycolysis both at rest and following activation. These disturbances in T-cell metabolism are highly consistent with findings in chronic viral infections and CD8 T-cell clonal exhaustion (McLane et al., 2019).

CD8 T-cell dysfunction, or a state of clonal T-cell exhaustion, in turn can lead to difficulty in controlling many of the persistent pathogens that are already nearly universally present in adult humans, such as EBV, CMV and HHV6; as these viruses become under poorer control, reactivating more frequently, they may further contribute to the immune dysregulation. It is becoming increasingly recognized that immune system dysregulation of acute COVID-19 and Long COVID may enable reactivation of latent viruses already present in their systems (Lehner et al., 2020; Le Balc'h et al., 2020; Gold et al., 2021; Proal and VanElzakker, 2021). Early studies and case histories demonstrate herpes virus reactivation in some acute COVID-19 patients (Le Balc'h et al., 2020; Garcia-Martinez et al., 2020). ME/CFS patients have been reported to have increased levels of antibodies to persistent viruses in the herpes family, like EBV, HHV6, CMV, VZV and HSV-1; treatment with antivirals, such as acyclovir and valacyclovir and valganciclovir, has had success in some patients (Henderson, 2014; Watt et al., 2012). There are also recent reports of finding herpes viruses by PCR in tissues like the brain (Kasimir et al., 2022). Patients treated with anti-TNF have also been reported to develop shingles (symptomatic VZV reactivation), requiring treatment, and have been found to have EBV and CMV by PCR in the stool (Kim and Solomon, 2010; Santella et al., 2022). In fact, other T-cell mediated autoimmune diseases, such as inflammatory bowel disease has been associated with reactivation of herpes viruses, as EBV has been found in the mucosal lining of the gut in some of these patients (Zhang et al., 2022; Wang et al., 2022). Dysregulated immune responses to EBV have also been strongly linked to the immunopathogenesis of multiple sclerosis, as well as finding evidence of EBV in the brain (Pender et al., 2017, 2018). Symptomatic herpes virus reactivation, such as VZV, has been previously reported following vaccination (Walter et al., 1999). It would, therefore, not be too surprising that ME patient 1, who did not produce any TNFα from her CD8 T-cells prior to vaccination, might develop chronic shingles (VZV) and cold sores (HSV-1) following vaccination.

It is likely other pathogens known to persist in humans, including RNA viruses such as enteroviruses, or bacteria such as M. pneumonia, or fungi such as C. albicans, that are controlled by CD8 T-cell responses may also be reactivated and be contributing to T-cell dysfunction. Identifying which persistent pathogens are reactivated in any one patient and determining if they need to be treated will be challenging, but possible. The nebulized agent described here has anti-viral, anti-bacterial and anti-fungal properties. It is thus useful to help treat multiple pathogens at the same time. However, anti-pathogen drugs such as anti-viral or antibiotic medications may be needed in addition to this nebulized therapy as seen in some of the patients in this study.

In patients with chronic ME/CFS, studies suggest that subsequent new infections and antigenic challenges, such as with vaccines, may exacerbate their symptoms (Beyond Myalgic Encephalomyelitis, 2015). In the case of ME patient 1, viral infections or the SARS-CoV-2 vaccine appeared to trigger all 4 major relapses of severe ME/CFS over the course of decades. Some ME/CFS patients have been reported to have exacerbation of their symptoms following SARS-CoV-2 vaccination (ANZMES, 2021; Johnson, 2022; Johnson, 2023). A recent study shows that people infected with SARS-CoV-2 prior to vaccination produced spike-specific CD8 T-cells at considerably lower levels—and with more dysfunctional response—than vaccinated people who had never been infected (Gao et al., 2023). Similar to our results, these findings suggest that SARS-CoV-2 infection damages the CD8 T-cell response, an effect similar to that observed in previous studies showing long-term damage to the immune system after infection with hepatitis C or HIV1, both persistent viruses that can induce T-cell clonal exhaustion (McLane et al., 2019). Unfortunately, established clinical testing by serology, and PCR of serum/plasma for viremia, is limited, making it difficult to determine if there is impairment in immune system control over existing persistent herpesviruses. It is likely that compensatory immune mechanisms keep some patients from becoming markedly viremic, so the presence of virus in peripheral blood or circulating immune cells, if any, may not be picked up by PCR.

Recent studies also suggest that reactivation of persistent viruses may be difficult to detect as they mostly reside within tissues, such as muscle, or even in the gastrointestinal (GI) tract and/or in stool, as seen in patients immunosuppressed by anti-TNF therapy (Santella et al., 2022; Solomon et al., 2022). SARS-CoV-2 is also thought to linger in the body such as the GI tract for long periods after initial infection, particularly in the neurons of the myenteric plexus (Gray-Rodriguez et al., 2022; Peluso et al., 2023). CMV is known to infect endothelial cells lining the vasculature (Takatsuka et al., 2003) and thus could potentially be replicating throughout the body, including the blood vessels in the brain. Currently, clinicians will need to maintain a high index of suspicion regarding reactivation of persistent agents and may find it useful to perform the herpesvirus serology panel outlined here. If the patient harbors IgM antibodies targeting any of these persistent viruses, this suggests reactivation, as in LC patient 1, or has high titers and clinical symptoms of chronic shingles or recurrent cold sores, as in ME patient 1, then the patient should be treated with the appropriate antivirals. In these two patients, introducing antivirals at 11–12 months into treatment with the nebulized agent (at which time overall severity scores had already decreased from 10 to 4–5) may have contributed to greater clinical improvement, such that overall severity scores decreased further (e.g., scores dropping from 4 to 5 to 2).

Other clinical factors may need to be considered before embarking on added anti-viral treatment if high titers are found for antibodies specific for multiple herpesviruses. Nonetheless, high antibody titers even against multiple herpesviruses at the same time, are strongly suggestive of significant CD8 T-cell impairment and potentially T-cell clonal exhaustion leading to difficulties controlling persistent infections. In the present retrospective study, only 3 of 8 patients were also treated with anti-viral therapy while on this nebulized treatment. Larger, controlled clinical trials are needed to determine whether adjunct treatment with antivirals during treatment with this nebulized agent could further enhance therapeutic outcome resulting in more rapid and permanent reversal of CD8 T-cell dysfunction, with greater control over persistent pathogens and more rapid improvement of symptoms.

Using functional ICS assays to examine CD8 T-cell cytokine production led to identification of CD8 T-cell dysfunction as a possible underlying immunopathologic pathway in some patients with ME/CFS and Long COVID. This type of assay may prove useful as a diagnostic tool and biomarker to track disease outcome during therapy. It is a relatively simple and well-established technology that has the potential to be adapted for use by clinical labs. Although altered inflammatory cytokines in ME/CFS patient serum and plasma have been previously reported to indicate significant immune dysregulation, circadian rhythms, sex and other individual differences, and various stressors can introduce variance. Measuring serum cytokine levels also is not generally useful as biomarkers, as serum cytokines are generally highly labile, with very short half-lives often due to the fact that other immune cells consume them rapidly and they are also very sensitive to how they are handled in the lab (Hornig et al., 2015; Broderick et al., 2010; Russell et al., 2016; Fletcher et al., 2009; Hardcastle et al., 2015; Maes et al., 2012a, 2012b). This can often lead to differing results between studies. Because one cannot identify which immune cell subsets are producing (or failing to produce) these immune molecules, these peripheral immune signatures also do not point clearly to the underlying immunopathogenic mechanisms (Hornig et al., 2015; Broderick et al., 2010; Russell et al., 2016; Fletcher et al., 2009; Hardcastle et al., 2015; Maes et al., 2012a, 2012b). Single cell RNA sequencing assays on multiple immune cells at the same time may someday be useful for diagnostic assays, but at present are still more of a research tool.

This is only the beginning of our research into immune dysregulation and its role in the immunopathogenesis of ME/CFS and Long COVID. In our ongoing research there appears to be evidence of other compensatory immune mechanisms for control of persistent pathogens in these patient populations. At present, we have demonstrated the utility of a CD8 T-cell functional assay for IFNγ and TNFα production following stimulation with a generic T-cell stimulus as a first logical and clinically feasible step in assessing CD8 T-cell dysfunction for diagnosis and tracking of responses to therapy. With further research we hope to be able to identify treatment-responsive subsets of patients, predict outcome and severity and be better able to understand the pathogenesis of ME/CFS and Long COVID and the mechanism of action of this nebulized antioxidant, anti-pathogen, and potentially immunomodulatoryagent in treatment of these disabling disorders.

Funding

This study was funded by NIH R01 AI159314-01 (LKS), Ramsey Award (SolveME, Inc.) (LKS), private donations (LKS) and Patient -Led Research Collaborative (LSK).

CRediT authorship contribution statement

Anna Gil: Conceptualization, Formal analysis, Investigation, Methodology, Project administration. George E. Hoag: Formal analysis, Writing – review & editing. John P. Salerno: Project administration, Resources. Mady Hornig: Writing – review & editing, Validation. Nancy Klimas: Validation, Writing – review & editing. Liisa K. Selin: Conceptualization, Formal analysis, Funding acquisition, Investigation.

Declaration of competing interest

The authors have conflict of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.