COVID-19 Antibody Testing: S vs. N Protein

Throughout the COVID-19 pandemic, researchers have been working tirelessly to understand the human immune response to SARS-CoV-2, including the duration and level of protection that antibodies may provide against re-infection.

Several tests have already been developed which can detect antibodies against SARS-CoV-2 and identify individuals who have experienced prior infection with the virus. However, findings from a study recently published in JCI Insight suggest that the information which can be inferred from a positive antibody test may depend on the specific antibodies that the test detects. Some detect the presence of antibodies against the spike (S) protein, while others detect antibodies against the nucleocapsid (N) protein.

Technology Networks spoke to Dr Iswariya Venkataraman, Scientific Affairs Lead at EUROIMMUN US, to learn about the differences between the S and N proteins and what makes them suitable as target antigens in serology testing. Iswariya also discussed the findings and implications from the study, which cautioned against the extensive use of tests that detect antibodies against the N protein only.

Anna MacDonald (AM): Can you provide our readers with an overview of serology tests, what they detect, and how this information can be used?

Iswariya Venkataraman (IV): Once there is contact with SARS-CoV-2, the immune system produces antibodies against this virus. Serological testing helps to understand the immune responses to SARS-CoV-2 in a dynamic qualitative manner and to identify individuals who were exposed to the virus.

Anti-SARS-CoV-2 antibodies can be detected in blood as early as 10 days after the onset of clinical symptoms. Therefore, these tests cannot be used to detect acute infections. However, detection of antibodies against SARS-CoV-2 virus complements viral testing. Antibody detection in combination with RT-PCR expands the detection window of SARS-CoV-2 infection and minimizes false-negative RT-PCR testing.

Serological assays are helpful in conducting epidemiologic studies to evaluate the extent of virus spread in communities and to determine infection fatality rate. Furthermore, these assays can identify individuals who can be donors of convalescent plasma to treat infected individuals. Lastly, antibody detection can help select appropriate individuals for clinical trials for vaccine or therapy development.

AM: In the case of SARS-CoV-2, most serology tests are based on detecting antibodies against either the N protein or the S protein. Can you explain the differences between these two proteins and what makes them suitable as target antigens?

IV: Coronavirus has four main structural proteins: nucleocapsid (N), spike (S), membrane (M) and envelope (E). The S protein consists of the S1 and S2 subunits. The S protein is highly immunogenic since it is located on the surface of the virus.

The N protein plays an important role in the transcription and replication of viral RNA, packaging the encapsidated genome into virions and inhibits the cell cycle process of the host cells. The N protein is abundantly expressed during infections and also has high immunogenic activity. Therefore, both N and S protein could be potential targets for the antibody-based detection of SARS-CoV-2. However, the N protein homology between SARS-CoV-2 and SARSCoV-1 is 90 percent, compared with the S protein (77 percent), especially the S1 subunit including the RBD (66 percent).

S1 subunit contains immunologically crucial receptor binding domain (RBD), which is the key target of neutralizing antibodies.

S1 subunit also has evolutionary low protein homologies within the coronavirus family suggesting that it could potentially demonstrate less cross-reactivities among the endemic coronaviruses. Recent studies have shown the N protein-based antibody assays could exhibit a higher false-negative rate compared with the S1 subunit, and that S1 subunit purified from mammalian cells demonstrated the highest performance to distinguish COVID-19 patients from controls. Therefore, the S1 subunit could be the specific target antigen for detecting SARS-CoV-2 antibodies.

AM: Recent research has cautioned against the extensive use of tests that detect antibodies against the N protein only. Can you tell us more about the study and its findings?

IV: The presence of antibodies alone does not indicate that the person is immune against potential re-infection. It is important to determine whether the antibodies can confer protective immunity long-term, providing virus-neutralizing antibodies that block viral infection, and help in clearing viral infection.  Neutralizing antibodies primarily target S protein in coronaviruses, in particular the S1 subunit and the RBD contained within the S1 subunit, preventing viral entry into the host cell. Authors of a recent study conducted by the University of Texas MD Anderson Cancer Center concluded that serological testing using the S1 protein including the RBD is needed to identify individuals with neutralizing antibodies. Researchers warned against serology tests that only confirmed the presence of antibodies against N protein, which may mislead people to falsely believe that they have potential immunity against re-infection. In SARS-CoV-2, there is a heterogeneous IgG response to the S1-RBD and N proteins and these responses do not always correlate with each other. In this study, patients with antibodies to the N protein and not the S1-RBD, failed to exhibit neutralizing antibodies. Additionally, the neutralizing capacity was higher in patients with antibodies against the S1-RBD compared to N protein (86% versus 74%). Furthermore, the rate of healthy individuals positive for antibodies against the N protein was higher compared with the S1-RBD (3% versus 1%). Overall, this indicates that antibodies against the N protein portrays prior exposure to the SARS-CoV-2 or related viruses, and not necessarily is an indicator of presence of neutralizing antibodies.

Results of this study and others validates EUROIMMUN’s Anti-SARS-CoV-2 antibody assay, which is based on the S1 subunit of the SARS-CoV-2 spike protein. As discussed earlier, the S1 subunit displays the lowest homology to the other members of the coronavirus family and is the focus of vaccine development programs and discussions about possible immunity against re-infection from the SARS-CoV-2 virus.

Okba et al. 2020 compared the performance of different Anti-SARS-CoV-2 ELISAs with plaque reduction neutralization tests (PRNT). The strongest correlation was observed with the EUROIMMUN ELISAs and the neutralizing test, indicating the ability of EUROIMMUN assays to detect antibodies that are potentially neutralizing.

AM: What implications do the results of this study have for antibody testing and how the results of such tests are used during the pandemic?

IV: In general, antibody testing will help understand the extent of SARS-CoV-2 spread in the community. However, there is still further research needed to help understand how long the antibodies would last and if they are truly protective against re-infection. Specifically, it is important to understand the long-term kinetics of these antibodies. There is an emphasis on understanding the immune responses that results in protection and the duration of protection conferred in infected individuals.  This study reinforces our approach that’s focused on detecting antibodies against the S1 subunit, and to have an assay that can potentially detect neutralizing antibodies and answer questions about possible immunity against re-infection in the future. This study stresses that there is a heterogeneous antibody response to the two SARS-CoV-2 viral antigens. Additionally, the N protein binding antibodies do not correlate with S1-RBD binding antibodies or possess neutralizing abilities. It is important to be cautious when using serology tests that are based on the N protein for addressing questions that are related to determining potential COVID-19 immunity. Many companies have conducted millions of tests to detect antibodies against the N protein, potentially misleading the antibody positive individuals regarding their plausible long-term immunity. EUROIMMUN was among the first companies to release an antibody assay based on the S1 subunit of the spike protein, which could be fully automated using their technologies. This assay was developed and validated in collaboration with leading institutions, reference laboratories and academic centers intimately involved in combating the COVID-19 pandemic. Additionally, EUROIMMUN’s IgG assay was the first and only FDA authorized antibody test, where the results of a new independent validation effort by the U.S. Government provided the scientific evidence used to support the emergency use authorization.

AM: EUROIMMUN offers tests detecting both S and N proteins. Can you explain the advantages of this offering?

IV: At EUROIMMUN, we also offer an ELISA* that is coated with a modified nucleocapsid protein (NCP). In this assay, we have removed unspecific epitopes from the full-length N protein enabling the NCP-based ELISA to detect specific antibodies to the SARS-CoV-2 virus. This assay could be helpful in discriminating a natural infection from that arising due to S1-based vaccination, indicating a potential role in vaccine studies. Additionally, combining assays for the detection of antibodies against S1 and NCP can increase confidence when assessing presence of antibodies against the SARS-CoV-2 virus. Both these assays could play an important role in assessing the immune status of an individual before and after vaccine administration.

* This device has not been FDA cleared or approved; This device has been authorized by FDA under an EUA for use by authorized laboratories; This device has been authorized only for the detection of SARS-CoV-2 IgG antibodies, not for any other viruses or pathogens; and, This device is only authorized for the duration of the declaration that circumstances exist justifying the authorization of emergency use of in vitro diagnostics for detection and/or diagnosis of COVID-19 under Section 564(b)(1) of the Act, 21 U.S.C 360bbb-3(b)(1), unless the authorization is terminated or revoked sooner.

Iswariya Venkataraman was speaking to Anna MacDonald, Science Writer for Technology Networks.