The patients with neuro long COVID have a distinct pattern of anti-nucleocapsid-specific immune responses

In this study, Australian authors examined the T cell and antibody responses to the SARS-CoV-2 nucleocapsid (N) protein to identify activation signatures which distinguish neuro-PASC patients from healthy COVID convalescents. Multimodal analyses revealed a distinct pattern of T cell activation and anti-nucleocapsid specific immune responses in neuro-PASC patients.

Neurologic manifestations of post-acute sequelae of SARS-CoV-2 infection” (neuro-PASC or neuro-long COVID) include headaches, visual and olfactory dysfunction, gait disturbances, paresthesia, coordination problems, and cognitive impairments. Their presence is associated with a long-term impairment of functional ability. Patients with mild acute COVID-19 presentation, who were not hospitalized for pneumonia or hypoxemia, often have these symptoms. Despite extensive mapping of the spectrum of neurological sequelae in patients with neuro long COVID, no significant progress has been made in comprehending the underlying mechanisms.

Although much attention has been focused on the spike (S) protein, many other SARS-CoV-2 proteins play equally critical roles in the viral life cycle. SARS-CoV-2 nucleocapsid (N) protein is the basis of viral RNA genome packaging, but it is also the most abundant protein in virions and a high immunogenicity antigen.

About the study

The research objective of this study was to identify and characterize T cell responses to SARS-CoV-2 which are linked to neuro-PASC pathogenesis, and to specify how these responses differ from COVID convalescent controls without symptoms of long COVID. The authors focused on a group of patients who had a mild acute COVID-19 infection, but later developed neuro-PASC.

The study included 94 patients with neuro-PASC, 44 healthy COVID convalescent controls, and 34 healthy controls. Neuro-PASC patients had PCR+ or seropositive IgG results for SARS-CoV-2. For more than 6 weeks following the acute SARS-CoV-2 infection, they experienced persistent symptoms, such as headaches, fatigue, brain fog, and myalgia. 77 of them were not hospitalized for pneumonia, and had a mild disease. The COVID convalescent controls had PCR+ or seropositive IgG results for SARS-CoV-2 before vaccination, and they did not experience neurological symptoms for more than 4 weeks after the acute SARS-CoV-2 infection. Healthy controls tested PCR- for SARS-CoV-2 and were also seronegative for IgG against SARS-CoV-2 spike receptor-binding domen prior to vaccination.

Before assaying T cells for non-spike responses, 30 subjects across all three groups were vaccinated with the Pfizer BNT162B2 or Moderna mRNA-1273 mRNA vaccines.

The second version of the National Institutes of Health Toolbox test which measures processing speed, attention, executive functions, and working memory was used to measure cognitive impairments in neuro-PASC patients. The PROMIS-57 questionnaire was used to assess quality of life. Neuro-PASC patients scored lower on physical function and higher on anxiety, depression, fatigue, sleep disturbance, and pain. The second version of the National Institutes of Health Toolbox test showed that neuro-PASC patients had lower scores in the attention domain, indicating cognitive dysfunction.

The findings further revealed a distinct immunological signature in patients with neuro-PASC compared to convalescent controls.

The CD4+T cells from neuro-PASC patients were more activated by N antigens, and they produced more tumor necrosis factor alpha (TNF-α) than COVID convalescent controls.

The CD8+T cell subsets, CD8+TEM and CD8+TEMRA cells from neuro-PASC patients were less activated by N antigens compared to COVID convalescent controls, despite higher percentages of CD8+TEMRA cells in neuro-PASC patients compared to control groups. Furthermore, high depression scores correlated with reduced CD8+TEM cell polyfunctionality. Researchers speculated that CD8+TEMRA cells may be functionally anergic in neuro-PASC patients compared to convalescent controls, and this finding may contribute to the pathogenesis of PASC.

In neuro-PASC patients, although CD8+memory T cells showed reduced activation by N antigens, they displayed enhanced production of interleukin (IL)-6 in response to N protein. Plasma samples from patients with neuro long COVID had higher levels of soluble IL-6 and IL6-receptor-subunit-beta than COVID convalescents. The increased severity of neurologic symptoms, including pain, was correlated with production of IL-6 by CD8+T cells.

Neuro-PASC patients also had an elevated titer of anti-N IgG compared to COVID convalescents and healthy controls.

Researchers also assessed the relationship between cognitive and psychiatric clinical measures and virus-specific immune responses. In patients with neuro-PASC, the production of interferon gamma (IFN-γ) specific for the C-terminal region of the SARS-CoV-2 N protein (N3) was negatively correlated with scores on cognitive tests. Higher N3-specific IFN-γ response was observed in patients with lower scores for attention and executive functions. At the same time, the production of IFN-γ specific for N3 was positively correlated with anxiety scores.

Also, the findings showed elevated levels of proteins associated with immunoregulatory pathways in patients with neuro-PASC, contrasting with elevated levels of proteins related to pro-inflammatory and antiviral pathways in convalescent control subjects. In patients with neuro long COVID, the severity of symptoms and cognitive scores were significantly correlated with the level of immunoregulatory proteins.

In conclusion, these data suggest wide-ranging changes in anti-nucleocapsid specific immune responses in neuro-PASC patients. This has important implications for appropriate diagnostic, prevention, and treatment strategies. The observed imbalance between immunoregulatory and antiviral pathways may play a role in the pathogenesis of neuro long COVID.

This article was published in Frontiers in Immunology.

Journal Reference

Visvabharathy L et al. Neuro-PASC is characterized by enhanced CD4+ and diminished CD8+ T cell responses to SARS-CoV-2 Nucleocapsid protein. Front Immunol, 29 May 2023. Sec. Viral Immunology. Volume 14, 2023. (Open Access)