SARS-CoV-2 and the Antibody Response in COVID-19 Patients

The human immune system consists of various cellular differentiation pathways, which play distinct functions in responding to the presence of foreign antigens and micro-organisms in the body. Initial responses to foreign pathogens, such as viruses or bacteria, typically result in an early innate immune response¹. A portion of this response is mediated by cells bearing specific pattern recognition receptors, which detect foreign antigens on the surface of a pathogen, released by the pathogen, or released through the interaction of the pathogen and host cells¹. If the innate response fails to resolve an infection, an adaptive immune response may be initiated, where white blood cells or lymphocytes, with receptors capable of binding and detecting a specific foreign antigen, will go through clonal amplification and further maturation processes¹. An important mediator of this response occurs via lymphocytic cells called B-cells, which characteristically mature in the bone marrow¹. During a primary innate immune response, naïve B-cells will recognize foreign antigens in the body. Along with additional stimuli, this activates the B-cells in order to further stimulate their differentiation into effector cells types such as plasma cells (Figure 1). Plasma cells secrete Y-shaped proteins, called antibodies, into the extracellular environment¹. B-cells may also further differentiate into memory B-cells, which may survive for decades in order to repeatedly produce an antibody-mediated response upon reinfection with a specific antigen¹. This process of B-cell maturation, production of plasma and memory B-cells, and secretion of specific antibodies is part of the humoral immune response¹.

During the humoral immune response, the secretion of antibodies by plasma cells into the extracellular environment may contribute to immunity in three ways. The first is through neutralization, through which an antibody will bind to an antigen on a foreign pathogen and block it from invading or attaching to a host cell, thus impeding further infection¹. Another method is through a process called opsonization, in which antibodies attach to a foreign pathogen and induce phagocytosis or engulfment by innate immune cells. This leads to degradation of the pathogen, thus removing it from the body¹. Lastly, antibodies may attach to a pathogen and activate the complement system, a very early innate immune pathway that assists in degradation of the pathogen¹.

The immune system produces and circulates antibodies, specifically IgM, following an acute phase of disease. This is referred to as seroconversion, and it indicates that a recovery or convalescent phase of disease has begun. Once seroconversion occurs, antibodies may be detectable in the blood by antibody tests, or serological assays. Serologic data derived from antibody subclass and titer provides a history of infection and can be used to determine the nature of an infection, monitor population-based prevalence of disease, assess disease stage, guide vaccine development and identify an infectious agent such as a virus^1,3. In addition, serologic data can be used to better understand the time course of an antibody mediated response to expand biological knowledge. This may be assessed through measuring titers of specific antibodies for different antigens and Ig classes over time using serological assays³. For example, in a typical immune response, IgM is generally detected during the first 3-14 days following initial infection³. IgG, IgA or IgE production may begin later, following a class switching event³. IgG and IgM concentrations will eventually peak and then either slowly decrease or plateau, in a time frame that could be specific to the pathogen and disease burden³. These assays can also be combined with other tests, which demonstrate the ability of the antibodies to neutralize virus³. Response to infectious disease agents may vary, so understanding the immune response for new and emerging pathogens is essential for determining the value and application of a serological test.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), formerly referred to as 2019-nCoV⁴, is the novel infectious disease agent which causes COVID-19. SARS-CoV-2 serological assays used to detect antibodies in the blood (ex: serum or plasma)⁵, can be useful for identifying exposed persons who have mounted an immune response but may be asymptomatic, show negative results in PCR testing, produce a low viral load, or present later in the course of disease. They may also be useful when respiratory sampling is not possible⁶. However, SARS-CoV-2 serological assays are not currently recommended for diagnosis or measurement of active infection in the United States (U.S.)⁶. According to the U.S. Food and Drug Administration (FDA), serological tests should only be ordered by physicians who are familiar with their use and limitations⁶.

The application of serological testing for COVID-19 disease is actively being studied. Other areas of potential use for SARS-CoV-2 serologic (antibody) testing include generating serologic data in order for researchers to study time-course of disease, supporting vaccine development strategies³, assessing the number of exposed individuals within a population⁷, assisting the medical community in understanding the adaptive immune response to SARS-CoV-2, or identifying eligible donors of convalescent plasma as a possible treatment strategy for critically ill COVID-19 patients^6,8.

The prevalence of SARS-CoV-2 infection in the U.S. population is currently unknown⁹. Serological assays, which detect the presence of antibodies to different viral proteins, may help assess prevalence of COVID-19 disease and determine whether an individual has been previously infected, but may not indicate whether an individual has developed immunity⁹.

Presently, numerous serology tests have been granted Emergency Use Authorization (EUA) by the FDA for SARS-CoV-2 antibody detection, which differ based on their performance characteristics⁹, the technological approaches they employ to detect antibodies and the class of antibodies they are capable of detecting in blood for different SARS-CoV-2 antigens^5,10. For example, some serological assays may only detect IgG, while others detect both IgG and IgM, or all antibody classes. They may also detect different SARS-CoV-2 viral antigens^5,10. Some tests have demonstrated greater sensitivity with combined IgG and IgM measurement^11-16, and recent meta-analysis demonstrates that using antigens to viral spike (S) versus nucleocapsid (N) proteins may also increase sensitivity of serological tests¹³. The complete utility of these tests has yet to be determined for COVID-19 disease, as the world continues to obtain new information regarding the immune response to SARS-CoV-2.

For additional information about SARS-CoV-2 immune response and studies, visit the Promega Connections blog: www.promegaconnections.com

What we know about the SARS-CoV-2 antibody-mediated immune response is derived from a limited number of recent studies which have collected serologic data from COVID-19 patients, using both experimental and commercially available serological assays. Most of those assays do not currently hold Emergency Use Authorization from the FDA in the United States. From these studies, it has been demonstrated that after 2 weeks post-disease onset, the average seroconversion rate is between 90-100% for IgM-IgG antibodies produced against the SARS-CoV-2 S and N proteins^14-19. (For more information on the SARS-CoV-2 viral structure and proteins please visit SARS-CoV-2 PCR and Serological Testing.) Serological data may be generated using several different types of assay technologies and formats, including antibody-mediated capture assays such as enzyme-linked immunosorbent assays (ELISA)^16, 20-23, as well as antigen-mediated capture approaches such as chemiluminescent immunoassays (CLIA)^{11, 19, 24-26}, immunofluorescence assays¹⁸, lateral flow assays²⁷, luciferase immunoprecipitation assays (LIPS)²⁸ and bioluminescent immunoassays. Using these types of serological assays, evidence of a humoral immune response in COVID-19 patients has been studied, and the profile of anti-SARS-CoV-2 antibodies in serum have been preliminarily described. Shown in Figure 3 is a graphical representation of the estimated antibody response in COVID-19 patients. Though many studies provide insights through preliminary serologic data, the question remains whether an antibody-mediated response may assist in clearing viral infection, and how clearance via antibodies might occur through various virus neutralizing immune responses.

There remains a limited understanding of the magnitude and duration of antibody responses in COVID-19 patients with different disease burden and how factors such as comorbidities, age and genetic background impact immune response; however, there is mounting evidence that higher levels (titers) of total anti-SARS-CoV-2 antibodies may be associated with higher disease burden when detected 2 weeks post-symptom onset^18,19,23-25. For example, Qu et al. demonstrated that critically ill patients in China produce higher titers of IgM and IgG antibodies to S and N proteins, and kinetically observed that IgM levels peaked later in disease course in COVID-19 patients who were critically ill versus patients with mild to moderate disease²⁴.

Similarly, Zhao et al. observed higher IgM and IgG titers in critically ill COVID-19 patients²³. However, this increase in serum antibodies did not coincide with a reduction in viral RNAs within nasopharyngeal samples of these critically ill patients²³. Moreover, disease burden in this case was not associated with the levels of SARS-CoV-2 IgM antibodies produced to the S protein or IgG antibodies produced to the N protein²³, suggesting that only certain antibodies may be associated with disease burden. In contrast, Long et al. found no association between IgG antibody production and clinical attributes of patients in China¹⁸, and Wӧlfel et al. demonstrate that in a small cohort of COVID-19 patients in Germany, the production of neutralizing antibodies in serum may not correlate well with clinical disease course²⁵. This result is in conflict with that of Ni et al. who discovered that COVID-19 patients recently discharged from a hospital setting produced significant titers of virus neutralizing antibodies against the receptor binding domain (RBD) of the S protein, which was associated with production of virally targeted lymphocytes¹². Likewise, an in vitro study demonstrated that antibodies against the SARS-CoV-2 S protein can inhibit entry into cells²⁹, and a few other studies have observed that COVID-19 patients produce neutralizing antibodies to the RBD domain of the SARS-CoV-2 S protein^30-35. Lastly, COVID-19 patients with severe acute infection have presented with higher plasma concentrations of pro-inflammatory cytokines, which contribute to disease-related mortality³². However, the ways that this attribute may impact the effectiveness of an antibody-response is currently unknown. Overall, there remains an unclear understanding of the relationship between the humoral immune response and COVID-19 disease, and as such it remains to be elucidated through further research which antigens and antibody classes may be associated with a more robust immune response, how that response occurs, and whether or not that response coincides with complete disease recovery in COVID-19 patients.

With the continuing development of technologies used for serological measurement of SARS-CoV-2 antibodies, the world may gain a better understanding of the emerging disease, COVID-19. This type of information is both useful to the medical community in the interim for monitoring patient response, and through research which has the potential to expand our knowledge of the body’s natural responses to the novel SARS-CoV-2 virus so that we may be able to discover new ways to control disease within the population.

Acknowledgments: Drafts of this article were edited by Kara Chamberlain, Ph.D. and Annette Burkhouse, Ph.D.