Coronavirus disease 2019 (COVID-19), a viral infectious disease that is caused by Severe Acute Respiratory Distress Syndrome Virus-2 (SARS-CoV-2) virus infection, has become a global pandemic following an initial outbreak in the People’s Republic of China at the end of 2019 and beginning of 2020. To date, more than 174 million people around the world have been infected with the virus and close to four million people have died of COVID-191,2. Upon infection, while most people will show no or mild symptoms, a significant portion of the population will develop moderate to severe symptoms that require hospitalization, aggressive treatments such as mechanical ventilation, intensive care, and some eventually succumb to death3–5. The mortality rate based on the number of confirmed positive cases varies from on average 2–3% to as high as ~ 10% in some countries1.
Extensive studies have found that the immune response in COVID-19 patients is rather complicated, varies significantly from person to person, and patients’ immune response dynamics have a direct association with their clinical outcome6–14. Of particular interest, patients who developed severe symptoms or died of COVID-19 have reduced number of T cells and their immune responses are skewed towards type 2, antibody-mediated immunity15–21. These patients are more likely to experience cytokine storm that can lead to uncontrolled inflammation of the body, tissue damage, and eventually organ failure. In contrast, asymptomatic patients or patients with mild symptoms appear to have stronger type 1, cell-mediated immunity than patients with more severe symptoms22. In the fight against infectious pathogens, especially intracellular pathogens such as viruses, type 1 immune response is the frontline defense against the pathogen from the immune system. If type 1 immune response fails to stop the infection, type 2 immune response is initiated, clearing the virus by producing virus-specific antibodies. Indeed, higher anti-virus antibody titers were found in patients with more severe symptoms compared to those with only mild symptoms17,18.
While type 1 immunity is dominated with cell-mediated immune responses and type 2 immunity leads primarily to antibody-mediated immune responses, both types are inter-connected through the humoral immune system, particularly, the immunoglobin G (IgG) subclasses. In humans, IgG1 and IgG3 are associated with type 1 response while IgG4 is linked to type 2 response23,24. In animals, bovine IgG2 is associated with type 1 and IgG1 is associated with type 2 immunity25. In murine models, IgG2a is a specific indicator of type 1 immunity and mouse IgG1 is a marker of type 2 immunity26. Under typical, healthy conditions, there is a balance between type 1 and type 2 immunity-associated IgG subclasses. Upon infection or other changes of physiological conditions such as pregnancy or parturition, such balance may be disturbed27,28. The ratio of IgG subclasses in blood serum or plasma is often used as a marker to evaluate the relative balance of type 1 and type 2 immune responses in humans and animals29–32.
We previously developed a rapid blood test, D2Dx™ immunity test, that can detect and monitor the humoral immune status and responses in laboratory and farm animals33− 37. The test uses a gold nanoparticle (AuNP) as a pseudo virus pathogen to probe the humoral immunity in a blood plasma or serum sample. The AuNP is designed to react with type 1 and type 2 immunity-associated IgG subclasses non-specifically, but differently. Upon interaction with type 1 immunity-associated IgG subclasses, such as human IgG1, IgG3, bovine IgG2 and murine IgG2a, the AuNPs will form large aggregates, leading to a strong color change of the AuNP solution (Fig. 1). This color change is detected using a handheld colorimeter as shown in Fig. 1. The AuNP probe can also interact with type 2-associated IgG subclass, i.e., IgG4 for human, IgG1 for bovine, and IgG1 for murine. However, these IgG subclasses will not lead to AuNP aggregate formation, hence, no color change of the assay solution.
To illustrate the principle of this test, we present here a kinetic study of the AuNP interaction with different IgG subclasses. Figure 2A is the reaction kinetics between the AuNP probe with IgG subclasses indicative of type 1 immunity, i.e., human IgG1 and IgG3, bovine IgG2, and mouse IgG2a, while Fig. 2B is the reaction kinetics between the AuNP probe and type 2 indicating subclasses, namely, bovine IgG1, human IgG4, mouse IgG1. The association of human IgG2, mouse IgG2b and mouse IgG3 in type 1 versus type 2 immunity is not as clearly understood as other IgG subclasses, but the results are presented here as well. As clearly demonstrated from the study, all type 1-related IgG subclasses, regardless if it is from human, bovine or murine sources, caused dramatic color change of the assay solution; while all IgG subclasses associated with type 2 immunity, led to almost no color change of the assay solution.
In a blood plasma or serum sample, different IgG subclasses will compete to interact with the AuNP probe, and the measured color change of the assay solution is a quantitative indication of the relative balance of type 1 and type 2 immunity. A high D2Dx™ test score is indicative of a stronger type 1 immunity and weaker type 2 immunity; while a low test score correlates to a weaker type 1 immunity and stronger type 2 immunity. It should be noted here that although the AuNP probe is designed to primarily react with the type 1 and type 2 immunity-associated IgG subclasses differently, other molecules and biochemicals, especially complement proteins are also involved in the interaction with the AuNP probe, as specifically proven in our published study33.
Previously, we have used this new test primarily to study and monitor the humoral immune status and type 1/type 2 immunity balance change in laboratory animal models and agricultural farm animals33− 36. Our studies show that the test can detect active immune response in animals upon virus infections, immune function development in neonatal animals, immune status change in dairy cows associated with pregnancy and parturition. Based on these animal studies, we hypothesize that the D2Dx™ immunity test may be able to detect the immune status change in COVID-19 patients. Furthermore, if the clinical severity of the COVID-19 patients is associated with the relative balance between the type 1 versus type 2 immunity, the test may be able to detect this difference and the result may be used to predict the clinical risk of the patients.