The infection with severe acute respiratory syndrome coronavirus type-2 (SARS-CoV-2) can lead to a new disease called long COVID-19 or post-acute COVID-19 syndrome (PACS). This syndrome can occur in various populations, including children and young adults and those who had only mild COVID-19. Even though the precise origin of chronic COVID is not fully understood, it is well-known that viral infections have a lasting effect on the immune system and can result in organ-specific, disease-typical post-infectious consequences. The Austrian authors in this study investigated humoral and cellular immune parameters in unvaccinated COVID-19 convalescents ten weeks and ten months after their initial infection with the SARS-CoV-2 Wuhan Hu-1 strain.
In an earlier study, the same research team found long-lasting effects of COVID-19 on the immune system 10 weeks after the infection, even in those who had mild COVID-19. A sustained reduction in neutrophil count was associated with T cell activation, as evidenced by increased HLA-DR+ (CD8+ T cells) and CD38+ (CD4+ and CD8+) expression. In addition, COVID-19 convalescents differed markedly from uninfected controls by significantly higher numbers of CD3+CD4+CD127+CD45RA– and CD3+CD8+CD45RO+CCR7– effector memory T–cells, CD19+IgM+CD38+ transitional B–cells, and plasmablasts (CD19+IgM−CD38+). In the present study, the researchers investigated the long-term effects of COVID-19 on the immune system in the same group of previously studied COVID-19 convalescents 10 months after the infection.
About the study
The authors compared the results of humoral and cellular immune parameters 10 weeks and 10 months after COVID-19 in unvaccinated convalescents. The age- and size-matched, uninfected control group that was negative for SARS-CoV-2 at the time of venipuncture and asymptomatic 10 weeks prior to blood donation served as a reference. For the period between the two visits, none of the COVID-19 convalescents had reported symptoms of COVID-19. In addition, as licensed vaccines became available later, all included COVID-19 convalescents were unvaccinated.
The frequencies of reported comorbidities were comparable in COVID-19 convalescents (66%) and the uninfected control group (65%). The highest prevalences in both groups were reported for allergic diseases, followed by cardiovascular diseases, metabolic diseases, and chronic lung diseases. All other comorbidities (diabetes mellitus, hematopoietic diseases, immunosuppressive conditions, liver, neurological, or renal diseases) were reported only by a few individuals (<10%).
The authors first assessed the concentrations of IgG antibodies specific for the SARS-CoV-2 spike (S) protein, receptor-binding domain protein (RBD), and nucleocapsid protein (NC) over 10 months. According to the waning pattern of anti-SARS-CoV-2 antibodies, all COVID-19 convalescents were divided into three groups. Group 1 was characterized by a small to moderate (≤50%) decline of antibodies specific for the NC protein and a variable decline of antibodies specific for the S protein. Group 2 was characterized by a strong decline of antibodies specific for the NC protein (>50%) but a small decline of antibodies specific for the S protein (≤40%), and group 3 showed a strong decline of antibodies specific for both NC (>50%) and S protein (>40%). There was a significant difference in the mean age between groups 1 and 3 (56.2 ± 11.5 vs. 45.9 ± 12.3).
Multiparametric flow cytometry assessed whole blood leukocyte subsets, with a special focus on T and B lymphocyte subpopulations. Additionally, levels of serum cytokines and T and natural killer (NK) cellular interferon (IFN)-γ were measured.
Results
132 patients diagnosed with COVID-19 between May and August 2020, when SARS-CoV-2 Wuhan Hu-1 was the only circulating virus strain, and 98 uninfected healthy controls were enrolled in this case-control study. COVID-19 was confirmed by RT-PCR and/or anti-SARS-CoV-2 antibody assay. The cohort was analyzed 10 weeks (77.8 ± 24.6 days) and 10 months (9.5 ± 0.8 months) after the SARS-CoV-2 infection.
Since the IgG antibodies specific for the SARS-CoV-2 antigens S protein, RBD, and NC were stable or even elevated in 26 subjects, this group was excluded from further analysis because of possible clinically silent infection. In the remaining 106 COVID-19 convalescents, IgG antibodies specific for the SARS-CoV-2 proteins significantly decreased between 10 weeks and 10 months after the infection.
At 10 weeks after the infection, concentrations of IgG antibodies specific for the S protein were below the cutoff value in only 3 of 106 (2.8%) COVID-19 convalescents, but after 10 months, this number increased to 19 (17.9%) convalescents. Anti-RBD IgG levels were below the cutoff value in 26 of 106 (24.5%) COVID-19 convalescents at 10 weeks, but after 10 months, this number increased to 86 (81.1%) convalescents. None of the 106 subjects was negative for nucleocapsid protein, neither 10 weeks nor 10 months after the infection. Strong declines of antibodies specific for the NC and S proteins were associated with younger age and lower numbers of CD3–CD56+ NK and CD19+CD27+ memory B cells.
Graphical Abstract
Abbreviations: CD, cluster of differentiation; COVID-19, coronavirus disease 2019; IL, interleukin; IFN, interferon; NC, nucleocapsid protein; NK, natural killer; RBD, receptor binding domain; RTE, recent thymic emigrants; S, spike protein; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.
Blood leukocyte subsets
After 10 weeks, COVID-19 convalescents had lower numbers of circulating neutrophils than uninfected healthy controls. After 10 months, total leukocyte numbers in COVID-19 convalescents were lower than those at 10 weeks, including neutrophils, monocytes, and lymphocytes. The changes in total leukocyte, lymphocyte, and granulocyte counts were similar for all groups, but monocyte counts were reduced in groups 2 and 3 but not in group 1.
T cells
At 10 weeks after COVID-19, a sustained reduction of neutrophil counts was accompanied by T cell activation, as evidenced by increased HLA-DR+ (CD8+ T cells) and CD38+ (CD4+ and CD8+) expression. According to the authors, these findings may be caused by the shedding of SARS-CoV-2 particles, the extensive tissue damage in severe COVID-19 cases, increased IL-4 serum levels found in most patients, or by the moderately elevated IL-17A levels typically seen in severe COVID-19 cases.
T cell activation observed after 10 weeks disappeared after 10 months, except in 10 patients who were hospitalized due to severe acute COVID-19, in whom HLA-DR+ and HLA-DR+CD38+ T cells remained elevated. These changes in T cell types were similar in all groups.
At 10 weeks after COVID-19 infection, the investigation of naïve and memory T cell populations revealed that numbers of most naïve T cells, defined as CD3+CD45RA+CD62L+CD31+ recent thymic emigrants, were similar in COVID-19 convalescents and healthy controls. However, these numbers were almost cut in half after 10 months. This affected the CD3+CD4+CD45RA+CD62L+CD31+ helper T cell subset more than the CD3+CD8+CD45RA+CD62L+CD31+ cytotoxic T cell subset. Notably, more circulating recent thymic emigrants were found in younger patients than in older ones.
At 10 months after COVID-19 infection, a decrease in circulating recent thymic emigrants was accompanied by an increase in absolute numbers of CD3+CD45RO+CCR7+ central memory CD4+ and CD8+ T cell subsets. Absolute numbers of overall CD45RO+ memory T cells were not increased at 10 months, but CD3+CD4+CD45RO+CCR7+ and CD3+CD8+CD45RO+CCR7+ central memory T cell subsets were increased in 55% and 68.4% of COVID convalescents, respectively. These changes of T cell subsets were similar in all groups of patients with different antibody waning pattern.
Both the increase in CD3+CD4+CD127+ effector memory T cells and the decrease in Foxp3+ CD3+CD4+CD127−CD25+ T regulatory cells observed after 10 weeks had the tendency to return to almost normal levels after 10 months. However, CD3+CD4+CD45RA+CD127+ cells remained elevated throughout the observation period. According to the authors, a persistent elevation of CD3+CD4+CD45RA+CD127+ T cells may reflect expanded CD4+TEMRA (terminal effector memory T-cells that re-express CD45RA) cells.
These findings showed that 10 months after the COVID-19, mainly depleted was the pool of recent thymic emigrants but not of central memory T cells. The changes of T cell subsets observed after 10 months, such as normalization of effector and Treg numbers, decline of recent thymic emigrants, and increase of central memory T cell numbers, were independent of the antibody waning pattern.
NK cells
CD56+CD3- NK cell numbers were significantly lower at 10 months than those at 10 weeks, and this drop was mainly attributable to groups 2 and 3.
B-cells
At 10 months after the infection, overall numbers of B cells declined in the entire COVID-19 study group, with a significant drop in groups 2 and 3 and a plateau in group 1. A similar decline in overall circulating CD19+CD21+CD27+ memory B cells was observed after 10 months due to a significant decrease of non-class switched CD19+IgD+CD27+ memory B cells. These reductions were accompanied by the normalization of CD19+IgM+CD38+ transitional B cells and CD19+IgM-CD38+ plasmablasts, which was similar in all groups. While groups 2 and 3 showed reduced non-class switched B cells, a significant reduction of class-switched memory B cells was exclusively seen in group 3. In contrast, a drop of CD5+ B cells was observed across all three groups.
These findings showed a decrease in non-class-switched CD19+IgD+CD27+ memory B cells in COVID-19 convalescents 10 months after the infection compared to healthy controls.
Serum cytokine levels
At 10 weeks after the infection, COVID-19 convalescents had higher levels of certain cytokines, which may be considered indicative of Th1-dominated inflammation (e.g., interleukin (IL)-1β, IL-8, and IL-12) than the uninfected control group. These alterations resolved 10 months after infection. In parallel to this resolution, Th2-dominated serum cytokine patterns emerged, as evidenced by increased IL-4 and IL-10 serum levels compared to those at 10 weeks after the infection.
IFN-γ levels were similar in COVID-19 convalescents and uninfected healthy controls 10 weeks after COVID-19, but they decreased significantly after 10 months. This was accompanied by a decrease in IL-21 levels, while the overall low IL-17 levels did not change significantly.
These results suggest that the cellular changes were associated with a shift from Th1 to Th2 cytokine pattern. Interestingly, the extent of this shift was dependent on the antibody waning pattern. Group 1 showed considerable IFN-γ serum levels after 10 weeks and low IL-4 levels, which increased during the observation period. This led to a moderate change in the IFN-γ/IL-4 ratio and was accompanied by an increased IL-10 level. In contrast, groups 2 and 3 showed an IFN-γ-dominated response after 10 weeks and considerable IL-4 levels, which reversed during the observation period. This led to the more dramatic changes in IFN-γ/IL-4 ratios in groups 2 and 3, accompanied by decreased numbers of CD19+CD27+ B memory cells and CD3–CD56+ NK cells. The authors noted that future studies should investigate whether the group differences in IFN-γ and NK cells described above are the consequences of, for example, „original antigenic sin“ due to infection with common cold coronavirus strains. Interestingly, almost all (93%) COVID-19 convalescents suffering from a post-COVID-19 condition had an inverted IFN-γ/IL-4 ratio and were more frequently stratified in group 3 than in groups 2 and 1. Accordingly, the authors speculated that the inversion of the IFN-γ/IL-4 ratio could be a biomarker for post-COVID-19 syndrome.
Conclusion
A follow-up study of immune parameters in unvaccinated COVID-19 convalescents 10 weeks and 10 months after a first and mostly mild infection with the Wuhan-strain Hu-1 showed a long-term reduction of innate and adaptive immune cells, associated with a shift from Th1 to Th2 cytokine pattern.
The authors claimed that these findings were unexpected. Instead of an activation and expansion of CD3+CD4+ and CD3+CD8+ effector memory cells and transitional B cells and plasmablasts, the results showed significantly reduced numbers of adaptive immune cells, including T cells (particularly CD3+CD45RA+CD62L+CD31+ recent thymic emigrants) and B cells (non-class-switched CD19+IgD+CD27+ memory B cells). These findings could explain an immunological mechanism of long-term post-COVID-19 sequelae.
Since all leukocyte lineages originate from pluripotent CD34+ hematopoietic stem cells in the bone marrow, the authors speculated that SARS-CoV-2 could infect these cells, which could explain the multi-lineage reduction observed after a certain time. Alternatively, SARS-CoV-2 may affect the bone marrow stromal microenvironment (mesenchymal progenitors, osteoblasts, fibroblasts, and endothelial cells).
This article was published in Allergy.
Journal Reference
Kratzer B, Gattinger P, Trapin D et al. Differential decline of SARS-CoV-2-specific antibody levels, innate and adaptive immune cells, and shift of Th1/inflammatory to Th2 serum cytokine levels long after first COVID-19. Allergy. 2024;79:2482–2501. https://onlinelibrary.wiley.com/doi/10.1111/all.16210
