High-performing multi-immunoassay for early diagnosis and prognosis of COVID-19

A new study published on the preprint server bioRxiv* in December 2020 indicates the ability of anti-SARS-CoV-2 antibody titers and time of induction to differentiate between severe and mild COVID-19 and could lead to the development of a prognostic biomarker for use early in the course of the illness.

As the pandemic shows no signs of receding any time soon, researchers are looking into the antiviral antibody response to diagnose the infection, predict the severity of illness, and understand the nature of the immunity it elicits. Preliminary evidence suggests that severe COVID-19 is linked to high antibody levels and B cell responses.

Study: Elevated SARS-CoV-2 Antibodies Distinguish Severe Disease in Early COVID-19 Infection. Image Credit: Corona Borealis Studio / Shutterstock
Study: Elevated SARS-CoV-2 Antibodies Distinguish Severe Disease in Early COVID-19 Infection. Image Credit: Corona Borealis Studio / Shutterstock

Unconventional Antibody Responses in COVID-19

In order to better understand this, the researchers behind the current study performed a multi-antigen immunoassay to uncover the diverse antibody responses and their correlation with clinical severity. They examined samples of COVID-19 plasma from 52 acutely infected patients and 69 convalescent patients, comparing these with 106 and 137 healthy adults from before and after the pandemic, respectively. They measured the titers of immunoglobulin M (IgM), immunoglobulin G (IgG), and immunoglobulin A (IgA) against the nucleocapsid (N), spike S1 subunit, receptor-binding domain (RBD), and S1 N-terminal domain (S1-NTD).

Antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection do not always follow the canonical pattern, where IgM antibodies are first produced, followed by IgG. Many patients showed a rise in IgM and IgG at the same time. Again, neutralization activity was correlated with anti-S1 RBD antibodies, but antibodies are also formed against the spike protein S1 and S2 subunits, the S1-NTD, and the N protein. Antigens that include epitopes that bind both neutralizing and non-neutralizing antibodies can be used in antibody-based diagnostics.

Earlier research indicated a rise in specific antibodies in severe disease compared to milder forms, but the degree of overlap between these categories made it useless for prognostic purposes.

High Predictive Value of Multi-Assay

The current study, therefore, began with the development of a sensitive antibody assay. Using this, the researchers found that a reliable diagnosis can be made in the acute phase (<30 days from symptom onset) by using the combined values for IgA, IgG, and IgM, or IgG values alone, against S1 and RBD antigens, and N antigen, to a slightly smaller extent.

Overall, in acute infection, IgA targeting the N, S1 and S1-RBD antigens were also potentially useful, as was IgM specific to S1 and S1-RBD. This indicates that IgA, IgG, or IgM could be of use in diagnosing infection early in the acute phase.

However, if the two patient classes are analyzed separately from day 6 to day 30 following symptom onset, patients with severe or critical illness have higher levels of anti-S1, anti-S1-RBD, and anti-N IgG, IgA, or combined IgG, IgA and IgM, compared to those with mild or moderate infections.

IgM values were poorly predictive of infection in the convalescent period, especially those targeting IgM S1-NTD and N. IgA had stronger predictive value for S1 and RBD. The highest diagnostic value was seen with IgG against N, S1 and S1-RBD, with the latter classes showing high specificity and sensitivity for patients in the convalescent period.

False Negatives due to High Titers

Again, severe or critical illness was associated with a robust prozone effect in the serum. There was a bell-shaped binding curve for both severe and critical patient groups, indicating the occurrence of false negatives for antibody interactions with N, S1 and RBD. To prevent this from occurring, the investigators used samples diluted 1:500 to ensure the inclusion of as many patients as possible from the mild/moderate group, with a low antibody titer, while minimizing the impact of the prozone effect in the severe or critical group.

Serum from severely/critically ill patients had high neutralizing capacity, being capable of 50% inhibition of ACE2 receptor binding activity even when diluted several hundred times. This was not observed in the mild-moderate group. Anti-S1 IgG titers were measured at titers up to 45 μg/mL and 100-400 μg/mL in the mild-moderate and severe-critical groups. There was no significant difference between measurements taken from serum or plasma.

Slower and Lower Kinetics in Mild Disease

Finally, mild or moderate illness was linked to a slower and lower response in the form of anti-N and anti-S1 IgG antibodies, relative to severe or critical illness. In severe illness, the antibody titer increased early and to a higher level for almost all isotypes and for all the antigens, except only IgM targeting S1-NTD. Both classes of patients had comparable anti-RBD IgG and neutralizing antibodies at 2-4 months.

In the convalescent stage, the antibodies in the mild-moderate group rise to almost that of the severe-critical group, which registers a slight fall. However, IgG, IgA, and IgM against N and S1 fall rapidly in the latter group, accounting for their lack of usefulness in convalescent stage diagnosis.

What are the Implications?

In short, diagnosis of infection in both acute and convalescent stages can be made using either the antibody combination or IgG alone, against RBD, confirmed by anti-N and anti-S1 titers. Differentiation of mild-moderate and severe-critical cases was best done by combined isotype measurements, or anti-RBD, anti-S1 or anti-N assays. The best in both classes was the anti-S1-RBD.

The high ASC expansion in severe/critical disease in early infection is linked to higher antibody levels, suggesting that the B cells and ASC have different sources in mild-moderate and severe-critical infections. The early response in the latter is vigorous and comprises mostly IgG and IgA, while in the former, it conforms to the classical model, taking 4-6 weeks to develop. Beginning with extrafollicular activation that proceeds to a transient IgM production, it then switches to germinal center activation, leading to memory B cell and plasma cell generation and specific, high-affinity isotype-switched antibodies.

However, extrafollicular reactions proceeding directly to the isotype-switched phase also occur in this infection, probably accounting for the high IgG and IgA production in severe-critical cases. The germinal centers are disrupted in these patients. Later antibody profiles will vary with the extent of perturbation and restoration of germinal center function.

The researchers comment, “The striking early response produced by severe/critical patients may enable early detection of the patients most likely to experience poor outcomes.” Secondly, they point out, “The virus-specific antibody kinetics vary depending on disease severity and we speculate that specific viral antigens will have implications for long-term humoral protection to SARS-CoV-2 re-infection.”

And finally, “This unexpected difference in the speed and magnitude of humoral responses may reflect important differences in the immune response and provide early signs for patients who might benefit from targeted immunomodulatory interventions.”

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Dr. Liji Thomas

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.


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