Scientists from Hong Kong Baptist University (HKBU) along with researchers from various research institutions in China have discovered that the plasma metabolite glycylproline (gly-pro) is responsible for the wanning antibodies in recovered COVID-19 patients.
The study findings were published in the peer reviewed journal: Proceedings of the National Academy of Sciences (PNAS).
The intraindividual variability in the production and decay of antibodies in response to the virus and vaccines of SARS-CoV-2 has been well-documented, but the mechanism underlying this heterogeneity is poorly understood. The present study conducted absolute quantification of both cytokines and metabolites in the plasma sample sets of convalescent COVID-19 patients with (the CA group) and without (the CO group) the virus-specific antibodies.
After recovery from the disease, the plasma metabolomes of both groups were highly similar to those from healthy persons, except for a few metabolite markers (Fig. 1E and SI Appendix, Fig. S2). However, the plasma inflammatory cytokines of these convalescent individuals still kept clear “memories” of the onset of COVID-19 (Fig. 1E).
The cytokines of antiviral function, such as IFNγ (34) and TNF (35), which were up-regulated in the blood of COVID-19 patients (14), were still higher in both groups of convalescents (Fig. 2 B and C and SI Appendix, Fig. S1). IL-6 has been associated with severe COVID-19 symptoms and a poor prognosis. However, in these convalescents, the level of IL-6 was as same as that in the healthy people (SI Appendix, Fig. S1E).
The lower level of IL-6R (SI Appendix, Fig. S1B) was likely related to the recovery of these patients. A similar case is the plasma levels of the TNF-α receptor TNF-RII. The increased soluble TNF-α receptor has been proposed as an indicator of higher intensive care unit mortality of COVID-19 (36).
Together with cytokine storm, the main cause of fatality in COVID-19 patients (14, 15), the lower levels of these cytokine receptors were possibly the reasons for their survival. As compared with the CA group, the even lower level of TNF-RII in the CO patients (SI Appendix, Fig. S1B) might be associated with a milder immune response and rapid fading of the virus-specific antibodies.
In particular, IL-12p40 has the most down-regulated level in the CO individuals compared with that in the CA and healthy groups (Fig. 2D). It is well known that IL-12p40 is a component of the bioactive cytokines IL-12 and IL-23, acting as a chemoattractant for macrophages and promoting the migration of dendritic cells for pathogenic inflammatory responses (37), indicating that inflammation may be associated with antibody immunity in convalescents against SARS-CoV-2 infection.
To further identify the biomarkers from the plasma samples of convalescent patients, the application of LASSO using MLR facilitated the determination of a minimal number of features that can distinguish the three groups of samples. The classification results from various machine-learning algorithms (SI Appendix, Fig. S8) demonstrated the great potential of these molecules in both basic research and clinical applications.
Among them, the six molecules that were selected to classify the CA and CO groups (Fig. 4C and Table 1), especially gly-pro, are of value in the prognosis of COVID-19 and prediction of vaccination efficiency (Fig. 4). It should be noted that we applied ANOVA and excluded the variables (i.e., cytokines or metabolites) that showed significant alterations with age and sex before LASSO feature selection.
These data processing steps were largely responsible for the discovery of these valuable biomarkers in the cohort of disparate composition. More importantly, the fact that no significant difference in the positive days of convalescent patients (Fig. 1D) further supports that the biomarkers (6 cytokines and 11 metabolites) can reveal the molecular characteristics in either the CA or CO individuals discriminating from healthy subjects upon SARS-CoV-2 infection.
Due to cytokine features as the biomarkers, we further focused on the metabolic regulation in antibody immunity by gly-pro, the most accumulated metabolite in the CO individuals (Fig. 3A). The antibody response assay in the mouse model has validated the role of gly-pro in the production of antibodies against the RBD domain of SARS-CoV-2 (Fig. 4).
Increasing gly-pro via exogenous supply or inhibiting its digesting enzyme with Cbz-pro treatment resulted in a lower SARS-CoV-2 RBD-specific antibody level. The pharmaceutical inhibition of DPP4, the producing enzyme of gly-pro, rescued the adverse impacts of gly-pro on antibody production (Fig. 4).
Further confirmation in GC B, Tfh, and plasma cells of the vaccinated mice indicated that gly-pro supplement can suppress antibody immunity whereas sitagliptin can at least partially counteract the inhibitory effects of gly-pro on RBD antibody production in mice, supporting our notion that DPP4 inhibitors might be the therapeutic potentials for maintaining neutralizing antibody levels.
DPP4 is an aminopeptidase expressed in various cell types and interacting with a wide range of proteins, including peptide hormones, chemokines, and immunomodulatory proteins (38). Due to its function of degradation incretins, DPP4 has been associated with diabetes and obesity, the most reported comorbidities linked to the severity of COVID-19 (39–41).
It also functions in the immune response by digesting chemokines such as CXCL10 (42) and by forming a complex with other immunoregulatory proteins like adenosine deaminase (43). This protein is a receptor of MERS-CoV, another coronavirus that can cause severe respiratory disease (26). Its role in the course of COVID-19 still attracts a lot of attention and needs to be clarified. On the one hand, previous studies have demonstrated that DPP4 is not essential for the cellular entry of SARS-CoV-2.
However, three clinical trials independently conducted in Italy (21) and Korea (22) consistently reported that the applications of DPP4 inhibitors significantly reduced the rates of severity and mortality of COVID-19 patients with type 2 diabetes. Regarding the multifunction of this enzyme, various possible functional routes from DPP4 or DPP4 inhibitors to COVID-19 have been proposed (38).
By demonstrating the role of gly-pro, one of the DPP4 products, in the production of SARS-CoV-2 antibodies, our results provide a possible mechanism underlying the clinical efficacy of DPP4 inhibitors in COVID-19 treatment. An increase in DPP4 has been associated with diabetes and DPP4 inhibition is a widely used strategy in the treatment of type 2 diabetes mellitus (44).
A recent vaccination study on diabetic patients reported lower SARS-CoV-2 IgG and neutralizing antibody titers in persons with type 2 diabetes (45). Thus, our results have also provided us deeper insights into how the comorbid condition of diabetes mellitus influences the severity of this infectious disease.
Furthermore, our finding showed that gly-pro could modulate the immune response to the SARS-CoV-2 RBD vaccine. This direction effect has provided a new target for intervention, which might help avoid the possible side effects of overweight and immune suppression caused by DPP4 inhibitors. It should be noted that no convalescent patients with diabetes were enrolled in our study, suggesting that gly-pro plays a critical role in antibody immunity against SARS-CoV-2 infection.
Besides gly-pro, only a small proportion of measured metabolites showed significant alterations between various groups of plasmas. The elevated AMP level was found in both the CA (1.4-fold) and CO (2.5-fold) groups (Fig. 3C), indicating an increase in energy demand in the recovered patients.
Accordingly, the intermediates of the citric acid cycle (TCA cycle), isocitrate and succinate, decreased in both the CA (−1.1- and −1.2-fold, respectively) and CO (−1.5- and −1.6-fold, respectively) groups (Fig. 3D and SI Appendix, Fig. S3). The reduced levels of plasma of acylcarnitines observed in both convalescent groups are consistent with this trend (Fig. 3B and SI Appendix, Fig. S3).
These data suggested that to meet this raised demand for energy the convalescents might up-regulate the fatty acid β-oxidation pathway, an alternative pathway for energy supply (46, 47). One of the best-known concepts in immunology is that the immune response activities, such as fever, generating new immune cells, and producing antibodies require high levels of energy.
Intriguingly, although both altered consistently, the changes of these molecules reached higher extents in the CO group, indicating a larger energy gap in the convalescents of fading antibodies. It is worth further study to clarify if the shortage in energy supply is a reason for the rapid decrease of antibody production.
Our results of the correlation between inflammatory factors and metabolites also support this speculation. The CO group had the largest number of inflammatory factor–metabolite pairs of strong correlation (Fig. 3K). The 13 metabolites correlated to one or more cytokines include carbon sources glucose and lactate and the intermediates of the central metabolic pathway such as F6P, G6P, xylose, R5P, fumarate, and malate.
The positive correlation between these metabolites possibly indicates that central metabolism is the rate-limited pathway for the production of these cytokines. Among the pairs, the only negatively correlated pair is glucose and IL-6 (Fig. 3K). Glucose is the main energy source of most cells, and IL-6 is one of the characteristic cytokines in COVID-19.
The negative correlation might result from the consumption of glucose by the immune response. Moreover, a recent study demonstrated that DPP4 inhibition increases the levels of inflammatory markers in the plasma of regular-chow-fed mice but not that of high-fat-fed ones (48), further supporting the importance of energy supply in the systematic immune response to COVID-19.
It has been noticed since the start of the pandemic that males are more vulnerable to COVID-19 than females in terms of both morbidity and mortality across all ages (49). This is genetically sound because some essential proteins in SARS-CoV-2 infections are encoded by the X chromosome genes. These genes include angiotensin-converting enzyme 2 (ACE2), a cell-entry receptor of the virus (50), and Toll-like receptor 7 (TLR7), a lung-abundant receptor recognizing viral RNA to produce type 1 interferon (51, 52).
The clinical samples of male and female COVID-19 patients have revealed that the males had higher levels of IL-8 and CCL5 in their plasma and change in some cytokines, such as TNFSF10 and IL-15, was associated with severity only in the female group (53). The sex difference has also been observed from our data of convalescent patients.
In addition, more sex difference has been quantified from the metabolites (SI Appendix, Fig. S4). The 30 metabolites of varying levels only in the CO group but not the H group are probably correlated with the sex difference in the immune response to SARS-CoV-2 (SI Appendix, Fig. S5). Overall, sex-specific antibody immunity against the infections of SARS-CoV-2 wild type as well as variants still requires further exploration.