Long-term sex differences in symptoms and immune profile in Long COVID

doi:10.21203/rs.3.rs-7642874/v1

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Long-term sex differences in symptoms and immune profile in Long COVID

https://doi.org/10.21203/rs.3.rs-7642874/v1

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Background

Long COVID (LC) is a post-infectious condition affecting millions worldwide, characterized by persistent multisystem symptoms. Women are disproportionately affected, reporting higher symptom burden, particularly neurocognitive and neurosensory complaints. While short-term immunopathology has been described, the long-term clinical course, immune dysregulation, and sex-specific underpinnings remain poorly understood.

Methods

We analyzed 34 participants experiencing symptoms from 9 months to 5 years post-SARS-CoV-2 infection, hereafter called persistent LC (pLC), alongside 26 SARS-CoV-2–infected controls without persistent symptoms. Clinical assessments, symptom inventories, comorbidity analysis, and work capacity evaluation were performed. Immune profiling included flow cytometry of CD4⁺ and CD8⁺ T cells, NK cells, and B cells, as well as quantification of plasma cytokines, soluble factors, and cytotoxic molecules, analyzed in a sex-disaggregated manner.

Results

Women with pLC exhibited higher symptom burden, particularly neurocognitive and neurosensory complaints, which increased with age and disease duration, whereas men showed no clear age- or duration-related patterns. Comorbidities, especially affecting endocrine, metabolic, and circulatory systems, were more frequent in women and correlated with symptom severity. Immune profiling revealed subtle but sex-specific differences: women had reduced CD8⁺ T cell cytotoxic function, lower NKG2D and granzyme K expression, increased sCD40L and sFAS, and decreased perforin, whereas men displayed elevated TNF-α. NK cell function, B cells, and humoral immunity remained largely intact. Over half of participants reported functional impairments affecting work capacity.

Conclusions

Persistent LC is characterized by sex-specific differences in symptom burden and immune profiles. Reduced cytotoxic CD8⁺ T cell function in women may contribute to viral persistence and neurological symptoms, whereas elevated inflammatory markers in men suggest distinct immune pathways. These findings highlight the need for sex- and duration-specific management strategies, the identification of biomarkers, and the development of personalized therapies targeting specific pLC endotypes. Understanding these mechanisms may inform therapeutic strategies for LC and other post-viral and chronic inflammatory syndromes.

Persistent Long COVID

sex differential immunity

symptom persistence

immune dysregulation

cellular and molecular signatures

Long COVID (LC) is a post-infectious condition affecting millions worldwide, marked by persistent, multisystem symptoms, with women disproportionately affected, particularly by neurocognitive and neurosensory complaints. Despite growing knowledge of short-term immune changes, the long-term clinical course, immune dysregulation, and sex-specific mechanisms remain poorly understood. In this study, we analysed 34 participants with persistent LC (pLC) experiencing symptoms from 9 months to 5 years post-SARS-CoV-2 infection and compared them with 26 SARS-CoV-2–infected controls without persistent symptoms. Participants underwent clinical assessment, symptom inventories, comorbidity analysis, and work capacity evaluation, while immune profiling included looking at specific immune cells and proteins in the blood. We looked into differences between men and women. We found that women with pLC experienced more symptoms than men, especially fatigue, difficulty concentrating, and memory problems, and these symptoms tended to worsen with age and length of illness. Women also had more underlying health conditions, particularly related to metabolism, neurology, and circulation, which may contribute to their symptoms. Regarding the immune system, women showed changes in immune cells that help fight viruses, which could explain lingering symptoms and vulnerability to neurological issues, while men showed higher levels of general inflammation. More than half of participants reported difficulties with daily activities and work. These findings highlight that LC affects people differently based on sex, illness duration, and health history and underline the need for tailored approaches to treatment, better understanding of the disease, and strategies to support daily functioning and quality of life for those affected.

Persistent Long COVID (pLC) disproportionately affects women, who exhibit higher symptom burden, especially neurocognitive and neurosensory complaints, increasing with age and disease duration.
Women with pLC have more comorbidities, particularly in endocrine, metabolic, and circulatory systems, which may exacerbate symptoms through chronic inflammation and organ dysfunction.
Immune profiling revealed sex-specific alterations: women display reduced CD8⁺ T cell cytotoxicity (lower granzyme K and NKG2D), increased sCD40L and sFAS, and decreased perforin, while men show elevated TNF-α.
Over half of patients reported functional impairments affecting work capacity, underscoring the socioeconomic burden of pLC.
It emphasizes the need for sex- and duration-specific management strategies for pLC.

Five years have passed since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and COVID-19 continues to pose a global health challenge through Long COVID (LC). The World Health Organization defines LC as a post-infectious condition with symptoms persisting for at least three months after COVID-19 onset, lasting a minimum of two months, and not explained by alternative diagnoses [1–4]. Globally, an estimated 65 million people are affected, with cases rising daily [5–7]. LC presents with a complex, multisystemic symptom profile, encompassing over 100 manifestations that range from mild to profoundly debilitating [8–12], including fatigue, cognitive impairment, respiratory and cardiovascular symptoms [13–21]. These symptoms often impair quality of life, limit daily functioning, and, in many cases, prevent patients from working [13, 22]. Although some recover, many individuals have experienced persistent symptoms since early 2020, with neurological sequelae such as brain fog and increased dementia risk reported to last for at least two years [23–31].

LC risk factors include female sex, older age, pre-existing comorbidities, ICU admission during acute COVID-19, lower socioeconomic status, and smoking [12, 13, 32, 33]. Biological sex strongly shapes LC presentation, mirroring other post-acute infection syndromes such as myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) [34, 35]. Unlike acute COVID-19, where men face higher severity and mortality [36–39], LC disproportionately affects women, who report greater fatigue, neurocognitive deficits, headaches, and neurological sequelae, whereas men show more endocrine dysfunction [12, 40]. The role of biological sex in LC prevalence, symptomology, and underlying mechanisms has only recently begun to be explored [41, 42]. Investigating the cellular and molecular drivers of these sex differences may reveal critical insights into LC pathophysiology and inform precision therapeutic strategies.

While the short-term pathophysiology of Long COVID within the first year post-diagnosis is increasingly understood [43–45], its long-term clinical course, immune alterations, and organ involvement remain largely unknown. Addressing this gap is critical, as LC can last two years or more [46, 47] and may, like ME/CFS, become a lifelong condition for some patients. To this end, we analyzed a cohort of 34 participants with persistent LC (pLC), experiencing symptoms from 9 months up to 5 years post-infection, and characterized their clinical and immunological profiles in a sex-disaggregated manner.

Experimental model and subject details

A total of 34 persistent Long COVID (pLC) patients and 26 controls were recruited between June 2023 and March 2025. All pLC patients underwent medical evaluations to exclude alternative medical aetiologies for their persistent symptoms. All participants were recruited at Medical Health Center USF Cuidar Saúde- Seixal. Demographic and clinical characteristics are detailed in Tables S1 and S2.

Blood samples were collected by venipuncture in EDTA tubes and were immediately processed. All participants signed the informed consent at the time of enrolment in the study and pLC patients completed the clinical survey provided by the Portuguese General Directorate of Health (Direção Geral da Saúde). The survey included questions about COVID-19 data, namely acute disease severity (non-hospitalized or hospitalized), SARS-CoV-2 re-infection, vaccination status (number of doses, date and vaccine brand), employment status post infection and a list of 48 symptoms possible symptoms possible to be reported by LC patients.

All procedures were approved by NOVA Medical School ethics committee (178/2024/CEFCM) and by ethics committee for health from ARSLVT (2508/CES/2023), in accordance with the provisions of the Declaration of Helsinki and the Good Clinical Practice guidelines of the International Conference on Harmonization.

Inclusion and Exclusion criteria

Inclusion criteria for pLC patients were age above 18 years old, previous positive PCR test for SARS-CoV-2 and persistent symptoms for 9 months after initial SARS-CoV-2 infection not attributed to any pre-existing comorbidity. Inclusion criteria for control group include age above 18 years and previous positive PCR test for SARS-CoV-2 with no active symptoms post infection.

Exclusion criteria for both groups include age below 18 years old and morbid obesity.

Blood sample processing

Blood samples were first centrifuged at 1000 x g for 10 minutes to separate the plasma that was carefully removed and stored at -80°C until further analysis. Peripheral blood mononuclear cells (PBMCs) were then isolated by density gradient centrifugation (Lymphosed, Biowest) [48–50] and cultured with RPMI supplemented with 10% FBS and 1% Antimycotic-Antibiotic, at 37°C with 5% of CO2.

Flow cytometry

PBMCs were stained with a fixable viability dye eFluor™ 506 (Invitrogen) and surface labelled with the following antibodies all from BioLegend: anti-CD3 (UCHT1), anti-CD4 (SK3), anti-CD8 (SK1), anti-CCR7 (G043H7), anti-CD45RA (HI100), anti-CD56 (HCD56), anti-NKG2D (1D11), anti-CD19 (SJ25C1), anti-CD27 (O323) and anti-CD38 (HIT2). Cells were washed, fixed with 1% PFA and permeabilized with Saponin. They were intracellular labelled with the following antibodies all from BioLegend: anti-Perforin (B-D48), anti-Granzyme B (GB11) and anti-Granzyme K (GM26E7). Cells were washed with FACS buffer and acquired in BD LSR Fortessa X-20 and analysed with FlowJo v10.7.3 software (Tree Star).

ELISA

Antibody binding to SARS-CoV-2 trimeric spike protein or Nucleocapsid was assessed by a previously described in-house ELISA assay [48, 49, 51] based on the protocol by Stadlbauer et al [52]. Briefly, 96-well plates (Nunc) were coated overnight at 4ºC with 0.5 µg/ml of trimeric Spike or Nucleocapsid. After blocking with 3% BSA diluted in 0.05% PBS-T, 1:50 diluted plasma was added and incubated for 1 h at room temperature. Plates were washed and incubated for 30 min at room temperature with 1:25,000 dilution of HRP-conjugated anti-human IgA, IgG and IgM antibodies (Abcam, ab97225/ab97215/ab97205). Plates were washed and incubated with TMB substrate (BioLegend), stopped by adding phosphoric acid (Sigma) and read at 450nm. Cut-off for plasma samples resulted from the mean of OD₄₅₀ values from negative controls plus 3 times the standard deviation. Endpoint titers were established using a 3-fold dilution series starting at 1:50 and ending at 1:109,350 and defines as the last dilution before the signal dropped below OD₄₅₀ of 0.15. This value was established using plasma from pre-pandemic samples collected from subjects not exposed to SARS-CoV-2. As previously described [51], in each assay we used 6 internal calibrators from 2 high-, 2 medium- and 2 low-antibody producers that had been diagnosed for COVID-19 through RT-PCR of nasopharyngeal and/or oropharyngeal swabs. As negative controls, we used pre-pandemic plasma samples collected prior to July 2019.

Luminex

Plasma samples were thawed and tested in the LegendPlex Human panel (BioLegend) to quantify the levels of TNF-α, IL-10, perforin, sCD40L, sFAS, Granzyme A and Granzyme B. The assay was performed according to manufactor’s instructions and was modified by using half of the amount of all reagents. All plasma samples were diluted 2X with assay buffer, and sample concentrations were calculated according to the dilution factor. Briefly, 12.5 µl of diluted plasma or standard, and 12.5 µl of mixed beads were added to each well and incubated for 2 hours. The plate (V-bottom 96 well plate) was washed twice with 100 µl of wash buffer. Samples and standards were incubated with 12.5 µl of detection antibody for 1 hour followed by an incubation of 30 minutes with 12.5 µl of Streptavidin-PE. The plate was washed once, and samples were resuspended in 75 µl of wash buffer. All incubation steps were performed at room temperature and protected from the light. Samples were acquired in a BD Accuri C6 plus (BD Biosciences) and analyzed with the Windows LegendPlex software (v8.0 BioLegend).

Statistical Analysis

Statistical analysis was performed using GraphPad Prism v9.00. First, we tested the normality of the data by using Shapiro Wilk (n < 6) or D’Agostino & Pearson (n > 6) normality tests, by checking skewness and kurtosis values and visual inspection of data. Then, if the samples followed a normal distribution, we chose the appropriate parametric test; otherwise, the non-parametric counterpart was chosen. In two groups comparisons: for unpaired data, Mann-Whitney test and the unpaired t test were used as indicated in figure legends. Spearman correlation test was used in correlation analysis as described in figure legends. p value was considered significant at *p < 0.05, **p < 0.01, ***p < 0.001.

Characterization of the study participants

Our study included 34 persistent Long COVID (pLC) patients recruited between June 2023 and March 2025, all of whom reported ongoing symptoms, including drowsiness, concentration difficulties, persistent fatigue, and insomnia, for at least 9 months. The cohort comprised 71% females and 29% males (Fig. 1A, Table S1), with sex designation assigned by the attending physician based on biological sex at birth. The median age was 53.5 years (interquartile range [IQR] 13), with 4 participants over 70 years of age (Fig. 1B, left). Seventy nine percent of patients were white and 21% were black. Only two participants required hospitalization for COVID-19, and one of these required supplemental oxygen (Table S1). The control group comprised 26 SARS-CoV-2–infected individuals without persistent Long COVID, recruited concurrently with pLC patients. The cohort was 73% female and 27% male, with a median age of 51.5 years (IQR 26) (Fig. 1A, Table S2).

The body mass index (BMI) ranged from 20 to 37.8 in pLC patients (median 27.6, IQR 5.8) and 24.1 (IQR 5.5) in controls, with no significant differences between groups or by sex (Fig. 1C–D). Most participants had a single SARS-CoV-2 infection, though pLC patients had more positive tests and a longer interval between primary infection and sample collection (Fig. 1E–F).

Regarding COVID-19 vaccination status, two pLC patients and two controls had not received any COVID-19 vaccine at the time of sample collection (Tables S1–S2). Among controls, the majority (42%) had received four doses, whereas most pLC patients had received two (29%) or three (26%) doses; no significant differences were observed between groups (Fig. 1G). The time elapsed between the first vaccine dose and sample collection was also similar across groups (Fig. 1H).

Women with persistent Long COVID have more underlying health conditions

Comorbidities can impair the body’s ability to fight infections, and several studies have reported a higher prevalence of LC in individuals with chronic conditions [12, 13, 53–55]. Pre-existing comorbidities may also contribute to LC through an associated molecular profile, in which elevated inflammation drives oxidative stress, tissue damage, and organ dysfunction, thereby exacerbating and prolonging symptoms [13]. To assess whether comorbidity burden differed in our cohort and whether it was linked to sex, we analyzed the number of comorbidities in each group. We observed that pLC patients had more comorbidities than controls (Fig. 2A). Sex-disaggregated analysis revealed that this difference was driven by women. Women with pLC had more comorbidities than female controls (Fig. 2B, left), whereas no differences were observed between men (Fig. 2B, right).

To analyze participant comorbidities, we classified them into 10 health system categories. Overall, the most common comorbidities in controls were of circulatory system (CS; hypertension 9 participants, 35%), and of the endocrine, metabolic, and nutritional system (EMNS; obesity 6 participants, 23% and overweight 6 participants, 23%) (Table S3). In pLC patients, comorbidities of the EMNS (obesity and lipid metabolism disorders, 14 patients each, 41%), CS (hypertension, 9 patients, 26%), and psychological conditions (Psy; depression, 8 patients, 24%) were the most prevalent (Table S3). A sex disaggregated analysis showed that the frequency of comorbidities is higher in control men than women in all the health system categories (Fig. 2C). Curiously, this difference was leveled in pLC patients (Fig. 2D), with women exhibiting an increase in the frequency of EMNS and Psy comorbidities (Fig. 2D).

Symptom burden is greater in women with persistent Long COVID.

LC symptoms significantly affect daily living, reducing quality of life and often causing disability [56–58]. We first assessed symptom burden by counting the number of self-reported symptoms per individual and found that women exhibited significantly higher symptom burden than men (Fig. 3A). In our cohort, the most frequently reported symptoms were drowsiness, concentration problems, and persistent fatigue (73%), followed by insomnia and forgetfulness (70%, 21 patients), dizziness (67%, 20 patients), and anxiety (63%, 19 patients) (Figure S1A, Table S4). Less frequent symptoms, reported by only two patients (7%), included peripheral edema, difficulty urinating, fainting, skin rash, and hallucinations (Figure S1A, Table S4).

To examine sex differences, symptoms were grouped by health system categories and compared between women and men (Figs. 3B, S1B). Women reported higher frequencies of neurocognitive and neurosensory symptoms, including loss of interest, behavioral changes, depressive mood, slowed movements, and altered taste or smell. Symptoms such as chest pain, nausea, and tremor were reported equally by both sexes (Fig. 3B).

We next evaluated the influence of time since pLC diagnosis and patient age on symptom frequency. Women in our cohort had longer disease duration, with up to 60 months since diagnosis, compared with men, whose maximum duration was 48 months. Women diagnosed 37–60 months prior exhibited the highest symptom burden, whereas men with a 2-year diagnosis reported the most symptoms (Figs. 3C–D). Symptom frequency in women was highest between 61 and 70 years, while younger women reported fewer symptoms; no clear age-related pattern was observed in men (Figs. 3E–F). Among the 26 pLC patients who responded, more than half (58%) reported negative impacts on work (Figure S1C), with 60% of these indicating a need to stop working entirely.

Overall, our results indicate that women with pLC experience a higher symptom burden than men, particularly for neurocognitive and neurosensory symptoms. In women, symptom frequency increases with longer disease duration and older age, whereas no clear age-related pattern is observed in men. pLC also substantially affects daily functioning, including work capacity.

CD8⁺ T cells display sex differential cytotoxic phenotype in pLC patients

Although T cells play a critical role in SARS-CoV-2 clearance and recovery from COVID-19 [59–63], their involvement in LC remains unclear [64, 65]. Some studies have reported T cell alterations, including exhausted T cells [42], reduced CD4⁺ and CD8⁺ memory cells [42, 66], and elevated PD-1 expression on central memory cells [67], whereas others found no differences in total CD4⁺ or CD8⁺ T cells or their memory compartments [64, 68, 69]. To investigate cellular immune changes in pLC, we phenotyped CD4⁺ and CD8⁺ T cells by flow cytometry and compared their profiles between controls and pLC patients. CD4⁺ T cell profiles, including memory subsets and activation markers, were also examined (Figures S2A, S3A–E). Consistent with previous reports [64, 68, 69], total CD4⁺ T cell levels and activation status were similar in controls and pLC patients (Figures S3A–B). Memory subsets, defined by CCR7 and CD45RA expression, were also comparable between groups, including T effector memory cells (CCR7⁻ CD45RA⁻, Figure S3C), T central memory cells (CCR7⁺ CD45RA⁻, Figure S3D), and T memory cells re-expressing RA (CCR7⁻ CD45RA⁺, Figure S3E).

To characterize the cytotoxic profile of CD8⁺ T cells, we assessed expression of granzyme B and K, perforin, and Natural Killer group 2 member D (NKG2D) by flow cytometry (Figure S2B). Given that sex can influence LC presentation (Fig. 3) and progression [41, 42, 70], we disaggregated our analysis by sex (Fig. 4A–E). The frequency of CD8⁺ T cells was slightly increased in the pLC group (Figure S4A), with sex-disaggregated analysis revealing that this increase was driven by men (Fig. 4A). Examination of the cytotoxic profile showed no sex differences in granzyme B (Fig. 4B) or perforin (Fig. 4D), and no significant differences were observed between controls and pLC patients (Figures S4B, S4D), consistent with previous reports [64, 68].

Interestingly, the production of granzyme K by CD8⁺ T cells was significantly decreased in pLC patients, a change driven primarily by women (Figures S4C, 4C). Similarly, NKG2D expression was reduced in pLC patients, with this reduction also attributable to women (Figures S4E, 4E).

Next, we examined Natural Killer (NK) cells, which play a critical role in innate antiviral defense and may become impaired during acute COVID-19, reducing their ability to eliminate infected cells [71]. However, their role in post-acute infection, and particularly in LC, remains unclear [66, 71]. First, we assessed overall NK cell (Fig S2C) frequency consistent with previous reports [66, 71], we found no differences between controls and pLC patients, [66, 71] nor between women and men (Figures S4F, 4F). As for the expression of granzyme B, granzyme K, perforin, and NKG2D (Figs. 4G–J, S4G–J) by NK cells, no significant differences were observed between controls and pLC patients for any of the cytotoxic markers (Figures S4G–J).

Lastly, we assessed humoral immunity, as antibodies generated after natural SARS-CoV-2 infection provide protection against reinfection [72, 73], even though the role of B cells in LC is still a subject of active research [74, 75]. No significant differences were observed in total B cells, memory B cells, or plasmablasts between groups (Figures S2D, S3F–H). Consistent with prior studies [22, 68, 76], we found no differences in IgA, IgG, or IgM antibodies against the Spike or Nucleocapsid proteins between controls and pLC patients (Figures S3I–N).

Our results indicate that pLC is associated with subtle, sex-specific alterations in CD8⁺ T cell cytotoxicity, particularly reduced granzyme K and NKG2D in women, while B cells, CD4⁺ T cells, NK cells, and humoral immunity remain largely unaffected. This highlights a potential role for CD8⁺ T cell dysfunction in female patients as a contributor to persistent LC pathophysiology.

Persistent Long COVID patients exhibit sex-specific differences in systemic inflammatory profiles.

Recent studies have sought to link specific markers of immune dysfunction and inflammation with LC [77–80]. Cytokines such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF-α), and interferon-gamma-inducible protein (IP-10) have been repeatedly associated with the condition [81–85]. To investigate systemic inflammation in pLC, we quantified plasma cytokines in a sex-disaggregated manner. Consistent with prior reports [82, 84, 85], TNF-α levels were elevated in pLC patients (Figure S5A). Although TNF-α was also increased in women with pLC compared to female controls, the elevation was more pronounced in men (Fig. 5A). In contrast, anti-inflammatory IL-10 levels did not differ significantly between groups (Figs. 5B, S5B). sCD40L, a marker linked to inflammation [86] and vascular risk [87], was elevated in pLC patient serum, with the increase predominantly observed in women (Figs. 5C, S5C). Soluble Fas (sFAS) has been detected in the serum of COVID-19 patients, with levels correlating with disease severity [88]. In our cohort, sFAS levels were modestly increased in pLC patients, driven primarily by women, who showed higher circulating sFAS than female controls (Figs. 5D, S5D). To examine the cytotoxic profile in pLC patients, we measured plasma concentrations of perforin and granzymes A and B. Serum perforin was reduced in pLC patients, significantly in women, whereas granzymes A and B levels remained unchanged across groups and sexes (Figs. 5E–G, S5E–G).

Next, we investigated whether the time elapsed between pLC diagnosis and sample collection influenced plasma levels of cytokines, soluble factors, or granzymes by performing correlation analyses (Figures S5H–M). Interestingly, TNF-α, IL-10, sCD40L, sFAS, and granzyme A showed no correlation with disease duration (Figures S5H–K, S5M). Perforin was the only factor displaying a positive correlation, with levels increasing in patients with longer disease duration, although the correlation was modest (Figure S5L).

Collectively, our results indicate that pLC is associated with a sex-differential inflammatory profile. TNF-α levels are elevated, particularly in men, while women show increased sCD40L and sFAS, alongside reduced perforin, further supporting a potential impairment of cytotoxic function (Fig. 4C). Other immune mediators, including IL-10 and granzymes A and B, remain unchanged.

LC is an emerging syndrome, affecting around 65 million people worldwide [6, 7]. The world has been confronted with new SARS-CoV-2 variants, leading to repeated infections [89] and conducting to a high prevalence of LC [90]. Due to LC heterogeneity, the plethora of relapsing and remitting symptoms [91] and multisystemic organ involvement [10, 11], the exact mechanisms underpinning LC pathophysiology and long-term prognosis remain unclear. Here, we sought to contribute to filling this knowledge gap, by studying a cohort of 34 patients with pLC. In our cohort, women experienced a higher symptom burden, particularly neurocognitive and neurosensory manifestations, alongside a greater comorbidity load, which increased with age and disease duration. Men, in contrast, displayed stronger systemic inflammatory signals, notably elevated TNF-α, but fewer symptoms overall. Immune profiling revealed sex-dependent cytotoxic alterations in CD8⁺ T cells, with women showing reduced granzyme K and NKG2D expression, increased sCD40L and sFAS, and decreased perforin, while other lymphocyte populations and humoral responses remained largely unaffected. These findings highlight distinct pathogenic mechanisms in men and women and underscore the need for sex-tailored approaches in the management and study of pLC.

The exact etiology of Long COVID (LC) remains unclear, but several risk factors, including female sex, age, comorbidities, and lower socioeconomic status, have been identified [12, 13, 32, 33]. Consistent with these associations, pLC patients in our cohort had a higher number of comorbidities than controls, with women showing the greatest burden. Co-morbidities affecting women were enriched in the endocrine, metabolic, psycological, and circulatory systems, which may exacerbate disease through sustained inflammation, oxidative stress, and organ dysfunction. These comorbidities likely contribute both to the onset and persistence of LC, in line with previous studies [12, 13, 53–55]. Symptom analysis revealed that women reported higher frequencies of neurocognitive and neurosensory complaints, including fatigue, drowsiness, concentration deficits, and altered taste or smell, consistent with prior observations [42, 92]. The pattern of neurocognitive and neurosensory symptoms resembles myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), a condition characterized by severe fatigue, post-exertional malaise, and cognitive impairment, with a female-to-male prevalence ratio of approximately 4:1 [93]. Viral infections, such as by Epstein-Barr virus, have been implicated in ME/CFS, and emerging evidence suggests that SARS-CoV-2 may similarly trigger ME/CFS in a subset of patients [94]. Moreover, symptom burden in women increased with both age and longer disease duration, whereas men showed no clear age- or duration-dependent patterns, emphasizing the need for sex- and duration-specific management strategies. Importantly, more than half of responding patients reported negative impacts on work, with many needing to stop working entirely, highlighting the substantial functional and socioeconomic burden of pLC.

Our immune profiling revealed subtle but sex-specific alterations in CD8⁺ T cell cytotoxicity in pLC patients. While total CD8⁺ T cell levels were slightly increased in men, women exhibited reduced granzyme K and NKG2D expression, indicating impaired cytotoxic function. The overall increase in CD8⁺ T cells in patients may reflect ongoing immune responses against a persistent SARS-CoV-2 reservoir in multiple organs, as these cells are essential for viral clearance [95]. The fact that the increase was driven primarily by men may help explain why male sex is not a major risk factor for developing LC. NKG2D, a key activating receptor on CD8⁺ T and NK cells, acts as a co-stimulatory molecule in CD8⁺ T cells, enhancing cytotoxicity and contributing to long-term memory formation [96, 97]. Blockade of NKG2D has been shown to impair memory CD8⁺ T cell development [98]. In our cohort, despite the higher total CD8⁺ T cell counts, pLC patients, particularly women, showed reduced NKG2D granzyme K expression, and perforin levels in the plasma. This reduction may compromise the ability of CD8⁺ T cells to clear infected cells, potentially contributing to viral persistence, and may also impair the formation of long-term memory T cells, increasing susceptibility to reinfection. Consistent with previous studies, we observed no differences in the frequency of CD4 + T cells or in their memory subsets [68, 69]. Yet, the specific role of these cells in pLC etiology is still to be clarified [64, 65], as other authors have found alterations in central memory and exhausted profile [42, 66, 67]. NK cells, critical components of the innate antiviral response, displayed no significant differences in overall frequency or cytotoxic marker expression (granzyme B, granzyme K, perforin, NKG2D) between pLC patients and controls, or between sexes. This suggests that, in contrast to CD8⁺ T cells, NK cell cytotoxicity is largely preserved in pLC. Similarly, humoral immunity appeared intact. No differences were observed in total B cells, memory B cells, plasmablasts, or in antibody responses (IgA, IgG, IgM) against SARS-CoV-2 Spike and Nucleocapsid proteins between pLC patients and controls. Indicating that pLC is not associated with major deficits in the B cell compartment or antibody-mediated immunity. These findings point to a potential role for CD8⁺ T cell dysfunction in the pathophysiology of pLC, particularly in female patients.

Systemic inflammatory markers also exhibited sex-specific patterns. TNF-α levels were elevated predominantly in men, whereas women showed increased sCD40L and sFAS alongside with reduced perforin. Unlike other chronic infection-associated conditions such as ME/CFS, where TNF-a declines after 2–3 years [99], TNF-α levels in pLC remained stable over disease duration. Soluble CD40L levels were elevated in pLC patients, reflecting patterns seen in autoimmune and inflammatory diseases, where sCD40L amplifies systemic inflammation [86, 100–102] and can disrupt the blood-brain barrier [102]. Similarly, increased sFAS, which can interfere with apoptosis and the clearance of damaged or infected cells [88], suggests ongoing immune activation in women and may further exacerbate chronic symptoms. Together, these observations indicate that pLC is associated with systemic inflammation and cytotoxic dysfunction, with sex-specific patterns that may underlie the female bias in symptom severity and neurological manifestations.

Understanding the mechanisms driving pLC will not only advance LC research but also provide valuable insights into other infection-associated chronic conditions, including ME/CFS and chronic Lyme disease. Our findings add to the growing evidence that sex-differential immunity plays a key role in shaping disease trajectories, highlighting the urgent need for tailored therapeutic strategies.

This study has several limitations. The pLC cohort was relatively small and lacked ethnic and racial diversity. In addition, baseline clinical parameters prior to acute infection were not available, limiting our ability to assess pre-existing differences. Finally, patients unable to attend healthcare consultations, due to severe illness, reduced mobility, or social exclusion, were not captured, which may have introduced selection bias. Despite these limitations, our study provides a detailed, sex-disaggregated characterization of pLC patients, combining clinical, immunological, and inflammatory profiling. The inclusion of a well-matched SARS-CoV-2–infected control group strengthens our comparative analyses. Moreover, by examining long-term pLC patients, some with symptoms persisting over four years, we offer unique insights into chronic disease mechanisms, immune dysfunction, and sex-specific differences that can inform future therapeutic strategies.

Perspectives and Significance

Our findings highlight the complex, sex-specific nature of pLC. Together, these results emphasize the need for sex- and duration-specific management strategies, the identification of robust biomarkers, and the development of personalized therapies targeting distinct pLC endotypes. Our results provide mechanistic insights into symptom persistence and immune dysregulation, highlighting women’s heightened vulnerability. Looking forward, these insights could inform the design of clinical trials by encouraging sex-stratified analyses and testing therapies that restore cytotoxic T cell function, including immunomodulatory or antiviral strategies.

Women exhibit heightened symptom burden, particularly neurocognitive and neurosensory complaints, which increase with age and disease duration, whereas men show no clear age- or duration-dependent patterns. Comorbidities, especially affecting endocrine, metabolic, and circulatory systems, likely exacerbate these symptoms through sustained inflammation and organ dysfunction. At the immune level, we observed subtle but meaningful sex differences: women with pLC display reduced CD8⁺ T cell cytotoxic function, lower NKG2D and granzyme K expression, increased sCD40L and sFAS, and decreased perforin, whereas men show elevated TNF-α levels. These molecular signatures may contribute to symptom persistence, immune dysregulation, and neurological manifestations in women. Importantly, over half of patients reported functional impairments affecting work capacity, underscoring the socioeconomic burden of pLC. By integrating clinical, immunological, and inflammatory profiling in a sex-disaggregated manner, this work identifies potential biomarkers, therapeutic targets, and emphasizes the importance of personalized, sex- and duration-specific management strategies.

Ethics approval and consent to participate

Consent for publication

All participants sign the informed consent for publication.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was funded by European Union’s Horizon Europe research and innovation action through the projects MPS_NOVA (Grant Agreement No. GA 101159729), by EVCA Grant (Agreement No. GA 101079264) to HS and by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) to JG. This work was also supported by Research Unit UID/04462: iNOVA4Health and by Associated Laboratory LS4FUTURE (LA/P/0087/2020) funded by Fundação para a Ciência e Tecnologia / Ministério da Educação, Ciência e Inovação”. HS is supported by FCT through work contract CEECIND/01049/2020 and by the Associated Laboratory LS4FUTURE (LA/P/0087/2020), JG is supported by Associated Laboratory LS4FUTURE (LA/P/0087/2020).

Authors' contributions

JF, JG, CC, MG and MIN designed and performed experiments and analyzed the data. JF enrolled the subjects and collected demographic data. JG wrote the first draft of the manuscript. HS conceptualized the study, designed experiments, analyzed the data, supervised the project and wrote the final version of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank all the participants and families who allowed us to use their data, donated samples and filled in the LC questionnaire by Direção Geral da Saúde, without whom this study would not have been possible. We extend our thanks to all the Medical Health Center USF Cuidar Saúde- Seixal doctors and nurses, for their invaluable support in recruiting participants and working with families. We would like to thank the Flow Cytometry Platform of Gulbenkian Institute for Molecular Medicine for their technical support. We thank Rita Teixeira for experimental support and Hugo Vicente Miranda for reagents.

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You are reading this latest preprint version

Long-term sex differences in symptoms and immune profile in Long COVID

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Plain English summary

Highlights

Background

Methods

Experimental model and subject details

Inclusion and Exclusion criteria

Blood sample processing

Flow cytometry

ELISA

Luminex

Statistical Analysis

Results

Characterization of the study participants

Women with persistent Long COVID have more underlying health conditions

CD8⁺ T cells display sex differential cytotoxic phenotype in pLC patients

Discussion

Perspectives and Significance

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Long-term sex differences in symptoms and immune profile in Long COVID

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Plain English summary

Highlights

Background

Methods

Experimental model and subject details

Inclusion and Exclusion criteria

Blood sample processing

Flow cytometry

ELISA

Luminex

Statistical Analysis

Results

Characterization of the study participants

Women with persistent Long COVID have more underlying health conditions

CD8+ T cells display sex differential cytotoxic phenotype in pLC patients

Discussion

Perspectives and Significance

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

CD8⁺ T cells display sex differential cytotoxic phenotype in pLC patients