We investigated whether specific clinical and immune response features triggered during COVID-19 impact the development of long COVID and whether anti-SARS-CoV-2 immune responses could be involved in the risk of or protection against the occurrence of specific LC symptoms.
Overall, we found no striking associations between anti-SARS-CoV-2 antibody or IFN-γ responses and LC development. However, we identified several associations suggesting risk or protection for different LC clinical phenotypes based on anti-SARS-CoV-2 antibody profiles. All associations indicating potential deleterious effects of anti-SARS-CoV-2 antibodies involved LC symptoms classified as general or other. In contrast, for vital organ-related LC symptoms, we found exclusively protective associations, particularly against cardiovascular, pulmonary, psychiatric, gastrointestinal, musculoskeletal and otorhinolaryngological symptoms.
Collectively, our data suggest that, following predominantly mild COVID-19, antibodies targeting diverse SARS-CoV-2 antigens play a protective role against vital organ-related LC symptoms, especially cardiovascular (16%) and gastrointestinal symptoms (9.6%), both of which were infrequent in our study.
In our cohort of primarily mild COVID-19 convalescents from the first wave of the pandemic, the prevalence of LC was 45%, with no association found with hospitalization during the acute phase. Although most reports indicate a higher prevalence following severe COVID-1938, our findings, along with those of others24, emphasize that LC can develop even after mild COVID-19. This highlights the mechanistic complexity involved in LC development, as well as the biological burden experienced during the acute phase of COVID-19. Since our study’s focused the recruitment during Brazil’s first wave (February–March 2020), early cases included primarily travelers and their contacts, resulting in a higher socioeconomic status (SES) cohort, also due to limited testing availability at that time. While this homogeneity strengthens internal validity by reducing acute-phase care disparities, it may limit generalizability to lower-SES populations who face greater comorbidity burdens and healthcare barriers39. Socioeconomic factors influence both immune responses and post-acute outcomes40, suggesting potential variability in LC manifestations across populations. Future studies should utilize public health systems and community-based recruitment for broader representation. Still, our findings raise important hypothesis baseline LC mechanisms in a controlled early-pandemic cohort.
We recognize the use of self-reported symptoms is a limitation of the study, which may be subject to recall bias. While common in pandemic research and necessary for remote data collection, it is true this approach lacks clinical validation. Notably, our finding that neurological symptoms predominated aligns with objective measures in other studies41,43, supporting our observations. Future work should combine patient-reported outcomes with clinical assessments and biomarker data to strengthen phenotype classification.
In concordance with other studies41, we found a greater number and duration of symptoms during acute COVID-19 associated with the development of LC. This likely reflects an impaired capacity to control viral replication and/or persistent inflammation affecting several organs and systems, particularly the lungs and the nervous system (CNS) in our study. In the LC group, we observed a higher frequency of pulmonary, neurologic and psychiatric symptoms during COVID-19. Indeed, both the lungs and the CNS suffer extensive damage during SARS-CoV-2 infection40,41, resulting in persistent inflammation and dysregulated immunological activation and repair mechanisms42,43. Moreover, SARS-CoV-2 detection in postmortem tissues indicates viral persistence, mostly reported in the lungs44, the CNS45 and the heart45. Notably, these organs are severely and persistently affected by COVID-1946, even following apparent clinical recovery44,47.
The contribution of biological burden intensity during COVID-19 to LC development involves immunological perturbations, including potentially deleterious anti-SARS-CoV-2 immune responses48. We found no differential anti-SARS-CoV-2 humoral or IFN-γ responses in the LC group. Despite limited and conflicting literature, LC has been associated with a decline in NP-specific interferon-γ-producing CD8 + T cells, suggesting that a sustained IFN-γ response may be protective49. In contrast, LC has also been associated with persistently elevated levels of type I and III interferons50, TNF, IP10, IL-1β, and IL-651. Our findings suggest that the anti-SARS-CoV-2 IFN-γ response, in early convalescence is unlikely to play a major role in promoting or preventing LC following mild COVID-19. However, we only studied IFN-γ, other cytokines may be relevant.
Nonetheless, we found differential correlations between the neutralizing capacity and anti-SARS-CoV-2 antibody levels when comparing LC and nLC participants. The exclusive strong correlations in nLC suggest that time synchronization between certain antigen-specific antibody levels and neutralization may play a role in limiting infection-induced biological distress, contributing to reestablishing homeostasis and preventing LC development52. These potentially protective correlations were noted only at T1 (IgA anti-NP) and T2 (IgG anti-RBD). Synchrony between neutralization and the levels of some anti-SARS-CoV-2 antibodies may be a relevant LC-preventive factor early in convalescence and during the six months following COVID-19. Considering that the NP is located outside the viral/host molecular binding region, in addition to the ability to neutralize viruses, other anti-NP antibody-mediated functions are likely relevant in preventing LC. Indeed, diverse antibody effector functions have been highlighted in COVID-1953 but not in LC. Importantly, SARS-CoV-2 NPs may accumulate in some cell types, as myenteric plexus neurons and megakaryocytes54, and bind to proinflammatory receptors, promoting inflammation and tissue damage55. Accordingly, anti-NP antibodies could play a protective role by promoting NP protein clearance from tissues, avoiding/limiting damage, in synchronic action with antibody neutralization.
Intriguingly, a strong positive correlation between neutralization and anti-NP was also associated with the risk for LC, also at T1, specifically for IgG rather than IgA. Notably, IgG anti-NP and the NP itself can induce IL-6 in vitro56. Thus, IgG but not IgA anti-NPs could favor sustained inflammation, contributing to LC development. Furthermore, high IgG anti-NP levels are correlated with increased nerve growth factor (NGF) levels57, and increased NGF is associated with sustained inflammation58, suggesting that this correlation may reflect a biological setting prone to chronic inflammation.
Both potentially protective and deleterious correlations between neutralizing capacity and anti-SARS-CoV-2 antibody levels/ involved all three antigens and all immunoglobulin isotypes. Our data indicate that in addition to their protective role in controlling infection, anti-SARS-CoV-2 antibodies may also have deleterious effects, as reported for anti-spike antibodies and the spike protein itself59, anti-S2 spike antibodies60 displaying particular glycosylation61, and for IgG anti-NP62. Certain IgG subclasses may mediate antibody-dependent enhancement (ADE), where antibodies facilitate viral entry into cells, potentially exacerbating disease severity64. Pro-inflammatory IgG responses, such as those involving afucosylated IgG, have also been associated with severe COVID-19 and cytokine storms48.
Our best multivariate analysis did not include antibody levels and revealed a model with approximately 80% accuracy in predicting LC development, based solely on grouped acute-phase symptoms. This accuracy is very similar to the value found for the model that included sex and age. Although significant sex and age differences have been reported regarding symptoms and severity of COVID-19 (reviewed by65 we did not observe a substantial impact. Our results are consistent with meta-nalysis data, indicating no significant impact of age, but they are discordant regarding the impact of sex66. Additionally, long COVID-19 has been linked to certain pre-existing medical comorbidities, such as diabetes and hypertension66 but only few participants of our cohort presented these conditions.
We found a protective effect for acute-phase gastrointestinal and cardiovascular symptoms, against developing LC, whereas otorhinolaryngological, pulmonary, musculoskeletal and other symptoms, in the acute phase, were associated with an increased risk. It is challenging to provide strong biological interpretations for these intriguing results, especially considering that the gastrointestinal and cardiovascular systems are potential reservoirs for SARS-CoV-267. Nonetheless, within the LC group, we detected several anti-SARS-CoV-2 antibody responses associated with protection against cardiovascular symptoms during LC. Moreover, cardiovascular and gastrointestinal symptoms were the least frequently observed LC symptoms (16% and 9.7%, respectively). In contrast, anti-SARS-CoV-2 immune responses triggered during acute infection appear insufficient to prevent neuropsychiatric LC symptoms, which were highly prevalent in our cohort (neuro: 95%, psychiatric: 55%).
Within the LC group, we detected differential anti-SARS-CoV-2 antibody responses associated with various LC symptoms. Overall, the detection frequency and/or levels of anti-SARS-CoV-2 antibodies and/or neutralizing capacity (VNT) were associated with both the risk of and protection against specific LC symptoms. All associations indicating potential deleterious effects of anti-SARS-CoV-2 antibodies were exclusively observed for LC symptoms involving non-vital organs (general and/or other symptoms). In contrast, for LC symptoms involving vital organs, we observed only associations suggesting protective effects of anti-SARS-CoV-2 antibodies, primarily against cardiovascular LC symptoms, at T1 and T2 and occasionally at T3. Specifically, cardiovascular, neurologic, psychiatric, pulmonary, gastrointestinal and musculoskeletal LC symptoms were associated with either lower levels or a lower detection frequency of certain anti-SARS-CoV-2 antibodies. Thus, their presence and/or higher levels could be beneficial in resolving or limiting the infection burden, protecting against damage to vital organs62.
It is interesting to connect these findings to our multivariate analysis results indicating that the occurrence of cardiovascular symptoms during COVID-19 is protective against development of LC. We suggest that anti-SARS-CoV-2 antibodies may contribute to the resolution of acute cardiovascular symptoms, likely providing later protection against cardiovascular LC symptoms. While our stratified analyses face sample size limitations, robustness testing revealed that unmeasured confounders would need to explain 7.6–18.1% of residual variance to nullify our observed associations—values substantially exceeding the original R2 contributions (0.12–17.8%). This suggests our models are particularly robust for otorhinolaryngological associations (robustness R2 = 18.1%) and gastrointestinal symptoms (11.4%). Nevertheless, we caution against overinterpreting subgroup findings with smaller effect sizes such as cardiovascular symptoms at 2.6%. Notably, a protective role against fatal outcomes was suggested for increased IgA and IgG anti-NP levels in patients with severe COVID-1968. These findings are significant, as cardiovascular involvement in severe COVID-19 can lead to fatal outcomes.
Another interesting finding was the protective association of antibody persistence at T3 with specific LC symptoms: IgM anti-NP against tachycardia and psychiatric symptoms and IgG anti-RBD against depression. The detection of these antibodies approximately 1 year after COVID-19 onset suggests that, even at a later timepoints, some antibodies may play a role in limiting or controlling central nervous system (CNS) distress. In contrast, the persistence of higher levels of neutralizing antibodies (> 1/160) was associated with the occurrence of general or other LC symptoms, suggesting that neutralization may also have excessive or deleterious effects in some tissues but not others, as previously reported68.
Although antibody-mediated protective mechanisms regarding LC phenotypes remain unclear, we highlight some potential connections involving specific SARS-CoV-2 antigens. NPs can form multiprotein complexes, aggregating with α-synuclein, possibly with amyloid fibrils55, and accumulate in the brain microvasculature69. If this accumulation is involved in psychiatric symptoms, anti-NP antibodies could potentially contribute to NP clearance, thereby limiting neuropsychiatric disturbances. Moreover, late detection of IgM anti-NP at T3 may indicate viral persistence, suggesting that these antibodies are likely beneficial in limiting virus-induced damage. This is plausible since the NP is an early-response antigen that induces the production of antibodies that decrease over time70. Notably, higher levels of IgM anti-NP at T1 or T2 were also associated with protection against cardiovascular LC symptoms.
Nevertheless, we still face many mechanistic unknowns71. It is challenging to interpret why the detection of IgA anti-NP at T1 is associated with protection against cardiovascular LC symptoms while simultaneously posing a risk for general and other symptoms. These distinct and seemingly opposing activities likely depend on various biological factors, including the phase of the disease process, antibody levels and potential cross-reactivity with self-antigens, which could favor pathological autoimmunity, or even the antibody ability to bind to the FcRIIB receptor (CD32)72, providing inhibitory signals, and downregulating inflammatory/effector functions. Additionally, the categorization of symptoms reflects the involvement of diverse tissues with varying sensitivities to viral tropism and its pathogenic effects, along with a complex array of self-antigens that may serve as potential targets for pathological autoimmunity.
Although our data reveal intriguing associations between anti-SARS-CoV-2 antibody profiles and LC phenotypes, we emphasize these findings are observational and mechanistic interpretations remain speculative. While antibodies may show dual roles, protective via viral clearance versus harmful via inflammation, the identified signatures—particularly IgA/IgG anti-NP links to cardiovascular protection—offer testable hypotheses. Future work on antibody functional activity such as ADCC and cytokine profiling and viral persistence measurements will contribute to understanding the diverse roles of anti-SARS-COV-2 antibodies in long covid.
Differential complex biological settings likely impact the predominance of protective or deleterious antibody effects and LC phenotypes. These factors include genetic and epigenetic backgrounds, past immunological experiences73, concomitant reactivation of other viral infections, as reported in LC74, and the beneficial effects of anti-SARS-CoV-2 vaccination5. It is important to note that the participants in our study were not previously vaccinated. Therefore, our findings may not be applicable to diverse populations worldwide, as exposure to various infections and multiple environmental challenges can influence immune system activity and, consequently, the capacity to manage SARS-CoV-2 infection and LC development.
Further underscoring the complexity of LC, several systemic immune profile perturbations may persist even six months following COVID-19, even in asymptomatic individuals recovered from severe COVID-19, indicating that homeostasis has not been reestablished75. This disturbed immunological profile supports the idea of some sort of silent long COVID, that may eventually manifest as critical clinical events, such as acute myocardial infarction or cerebral vascular accidents. These clinically silent biological perturbations may, at least in part, account for the excess number of deaths reported worldwide following the COVID-19 pandemic76.
Collectively, our data demonstrate that LC following predominantly mild COVID-19 presents diverse symptoms affecting major organ systems, including the central nervous, pulmonary and cardiovascular systems. We show that different system-category symptoms are differentially associated with anti-SARS-CoV-2 antibody profiles, both in early convalescence and later, during ongoing LC symptoms, indicating both potentially protective and deleterious effects of anti-SARS-CoV-2 antibodies.
We highlight that following predominantly mild COVID-19, diverse anti-SARS-CoV-2 antibody responses play important roles in protecting against vital organ-related LC symptoms, especially cardiovascular symptoms. Our data underscore the complexity of the potential involvement of anti-SARS-CoV-2 immune responses in either protecting against or contributing to the development of different LC phenotypes.
The complexity of long COVID emphasizes the need for investigations into potential mechanisms, integrating multiple biological features, as these factors are likely intertwined and may impact different clinical outcomes. A significant challenge ahead is to identify the major critical mechanisms involved in the LC and its phenotypes, as well as to develop strategies that promote or enhance the organism’s capacity to reorganize itself and reestablish homeostasis.
