The immunologic and virologic determinants of long COVID – COV-LT2-0041 [version 1; not peer reviewed]

Price, David ORCID: https://orcid.org/0000-0001-9416-2737 and Davies, Helen 2026. The immunologic and virologic determinants of long COVID – COV-LT2-0041 [version 1; not peer reviewed]. NIHR Open Research 6 , 178. 10.3310/nihropenres.1115363.1

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Abstract

Study Aims: Our project aimed to identify the pathological mechanisms that contribute to the development of long COVID. Background: Long COVID is clinically heterogeneous and comprises at least four distinct patterns, which can be classified in broad terms as follows: (i) symptoms that persist after resolution of acute COVID-19; (ii) new symptoms that appear after resolution of acute COVID-19; (iii) symptoms associated with a new health condition that arises after resolution of acute COVID-19; and (iv) symptoms associated with the exacerbation of a health condition that existed before the defining episode of acute COVID-19. The clinical spectrum associated with these patterns is diverse and often involves multiple systems, including ongoing respiratory problems, gastrointestinal disturbances, and neurological/psychological conditions, and in the absence of a confirmatory test, the diagnosis can only be made clinically. This task is complicated not only by the diversity of presentations, which can encompass several disease syndromes, otherwise known as “domains” or “phenotypes”, but also by the fact that symptoms typically fluctuate and commonly occur in temporal clusters, informally known as “crashes”. Observational research has identified two main symptom groups, one involving primarily the respiratory system (chest pain, cough, and shortness of breath) with attendant fatigue and headaches, and the other involving multiple systems, most commonly resulting in musculoskeletal (joint pain, myalgia, and reduced exercise tolerance) and neurological/ neuropsychiatric symptoms (anosmia, ageusia, amnesia, anxiety, “brain fog”, confusion, and impaired concentration). The aetiology of this heterogeneous disease is poorly understood but is thought to be multifactorial, speculatively involving an immunologic/inflammatory component. Indeed, current data suggest that tissue damage results from persistent inflammation, the mechanistic origins of which remain obscure. Accordingly, we set out to fill this knowledge gap using a variety of experimental approaches to profile the immune system and search for molecular signatures of disease, comparing healthy convalescent individuals with no residual symptoms after a defined episode of acute COVID-19 and age/ethnicity/infection/sex/vaccination-matched patients exhibiting residual symptoms consistent with a diagnosis of long COVID. Methods: Clinical assessments included a detailed history (noting vaccinations), physical examination, chest radiographs, exertional oxygen saturations, lung function tests, full blood counts with differentials and screening for active clotting diatheses, biochemical tests, including urea and electrolytes, liver enzymes, indicators of inflammation, such as C-reactive protein and ferritin, and lactate dehydrogenase, and screens for autoantibodies (e.g., ANA and ENA). Immunologic assessments were designed to obtain a comprehensive picture of the innate and adaptive immune systems (e.g., polychromatic flow cytometry and quantification of antibody titers against SARS-CoV-2 and associated measurements of neutralizing activity and ADCC) and the plasma proteome (e.g., O-LINK). Virologic assessments included plaque assays and qPCR. Results: We found no obvious differences in clinical parameters that distinguished healthy convalescent individuals from patients with long COVID. Markers of classical (C1s-C1INH complex), alternative (Ba, iC3b), and terminal complement pathway (C5a, TCC) activation were significantly elevated in patients with long COVID. These markers in combination had an ROC predictive power of 0.794. Other complement proteins and regulators were also quantitatively different between healthy convalescent individuals and patients with long COVID. Generalised linear modeling further revealed that a clinically tractable combination of just four of these markers, namely the activation fragments iC3b, TCC, Ba, and C5a, had an ROC predictive power of 0.785. Symptomatic disease was not consistently associated with quantitative differences in immune cell lineage composition or antiviral T cell immunity. Healthy convalescent individuals nonetheless exhibited higher titers of neutralizing antibodies against SARS-CoV-2 than patients with long COVID, and extensive phenotypic analyses revealed a subtle increase in the expression of some coinhibitory receptors, most notably PD-1 and TIM-3, among SARS-CoV-2 nonspike-specific CD8+ T cells in patients with long COVID. We also identified a plasma biomarker signature of disease linking breathlessness with apoptotic inflammatory networks centered on the hub protein CD40 and dysregulated pathways associated with lung injury, cell cycle progression, and platelet activation, which could potentially inform the diagnosis and treatment of long COVID. Other plasma biomarkers were identified that correlated with fatigue or mobility. No differences in autoantibody profiles were detected between healthy convalescent individuals and patients with long COVID, and SARS-CoV-2 was not detected in plasma samples from healthy convalescent individuals or patients with long COVID. Key Findings: Our key positive findings were: (i) activation of the alternative and terminal complement pathways is a predictive feature of long COVID; (ii) SARS-CoV-2-specific antibodies exhibit relatively impaired neutralization activity in patients with long COVID; and (iii) specific biomarkers in the plasma proteome correlate with specific symptoms in patients with long COVID. Conclusions: Our data suggest that long COVID is a systemic disease characterised by ongoing inflammation and suboptimal humoral immunity, potentially indicating persistent infection with SARS-CoV-2. We suggest that further studies of affected tissue sites are warranted to identify the source of these systemic perturbations and reveal key mechanistic insights into the origins of long COVID. Dissemination: Our work has been published in appropriate scientific journals accompanied by press releases to bring the key messages into the public domain. We also disseminate our findings regularly at conferences and meetings attended by journalists, members of the public, and other stakeholders beyond the scientific community. Expected influence/impact: We anticipate that our findings will help guide future studies into the precise mechanisms that underlie the pathology of long COVID. More immediately, our data suggest that complement dysregulation and the associated inflammatory response are potentially viable targets for therapeutic interventions designed to ameliorate symptoms and break the pathogenic cycle of disease. There are now several complement inhibitors in clinical use that could be repurposed to treat long COVID. On this basis, we propose that pilot trials are now warranted to test the efficacy of such drugs under close clinical supervision, even for a relatively short period of time, with the aim of disrupting the proinflammatory cycle and restoring a normal pattern of homeostasis in patients with long COVID. There may also be scope to develop more effective antibodies to fill the ”neutralization gap” that exists in patients with long COVID.

Item Type:	Article
Date Type:	Publication
Status:	In Press
Schools:	Schools > Medicine
Publisher:	Taylor and Francis
ISSN:	2633-4402
Date of First Compliant Deposit:	30 October 2026
Last Modified:	30 Oct 2026 15:00
URI:	https://orca.cardiff.ac.uk/id/eprint/190555

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