By Sally Robertson, B.Sc

The team says that as well as improving the sensitivity and specificity of serology tests for early diagnosis of current infection, the findings could aid the identification of previous infection, which is essential for determining the rate of infection spread.

They could also lay the foundations for identifying which antigens should be prioritized as targets in vaccine development.

The team’s antibody assays showed that three antigens on the virus allow specific and sensitive detection of antibody responses early on in the infection.

“Our data reports the most extensive landscape of antibody responses of COVID-19 patients reported to date,” writes Sophie Valkenburg (School of Public Health, The University of Hong Kong) and colleagues. “We report, for the first time, the detection of antibody responses directed against an extensive spectrum of the SARS-CoV-2 antigens.”

A pre-print version of the paper is available in medRxiv*, while the article undergoes peer review.

Understanding more about viral antigens
The urgent need for a vaccine that will protect people against SARS-CoV-2 infection and mitigate the COVID-19 pandemic has led researchers to ask important questions about fundamental aspects of the immune response. In particular, they would like to know which viral antigens are responsible for triggering antibody responses and what this may imply about long-term protection.

The spike protein found on the surface of SARS-CoV-2 enables the virus to bind to host cells and is an important target in the development of diagnostic tests and vaccines.

Studies have shown that patients infected with SARS-CoV-2 develop a neutralizing response to the S1 subunit of the Spike protein, which contains the host-cell receptor binding domain. However, one recent study found that 10 of 175 infected patients did not develop this neutralizing response, yet ELISA assays still identified binding antibodies.

“These instances of low or no antibody responses by traditional serological approaches may lead to an underestimation of asymptomatic and mild infection and threaten the success of a potential vaccine that targets the S1 alone,” writes the team.

“Therefore, a broader landscape of antibody responses to a range of viral proteins needs to be assessed to better detect the immunogenicity of SARS-CoV-2 infection to improve the understanding of pathogenesis and immunity.”

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Aside from the structural spike protein, the genome of SARS-CoV-2 also encodes for about 20 non-structural proteins.

The open reading frame (ORF) 1a/b encodes a large protein that is cleaved into 16 non-structural proteins (NSP1-16). Other ORFs may encode other proteins, but their functions are not known.

Now, Sophie Valkenburg and colleagues have used the Luciferase Immunoprecipitation System (LIPS) assay to compare antibody responses among infected versus uninfected individuals (controls) to 15 potential antigens on SARS-CoV2.

The antigens included four structural proteins (S, N, M, and E), the three spike protein subunits (S1, S2, S2’) seven ORFs (ORF3a, 3b, 6, 7a, 7b, 8 and 10) and one NSP within ORF1a/b (NSP1).

“The LIPS technique has previously been used to distinguish infected versus vaccinated cases for influenza due to the presence of antibodies against non-structural proteins, and to characterize human infections by zoonotic spillover from bat viruses,” explains the team.

What did the study find?
The researchers identified significant between-group differences in the strength of responses to antigens S, S1, and S2’, but these proteins did not demonstrate high sensitivity, especially in cases of early infection.

Of the eight ORFs, six induced antibody responses in infected patients, namely NSP1 (ORF1ab), ORF3a, ORF3b, ORF7a, ORF7b, and ORF8.

The researchers used cut-off values to calculate that three of these antigens – N, ORF3b, and ORF8 – were informative and sensitive enough to be used in diagnostic tests. When they combined these three antigens, they found they could correctly distinguish all infected patients from healthy controls with 100% sensitivity and specificity.

What are the implications of the study?
Valkenburg and team say a test that combines several antigens other than the spike protein could provide useful extra information for identifying SARS-CoV-2 exposure and for avoiding false negatives.

“We found that the single test detection of N, S, or ORF3b antibodies at early time-points (day 4 to day 14) results in a high proportion of false-negative results, while the minimal combination of N+ORF3b+ORF8 LIPS tests is highly sensitive and specific.”

Importantly, notes the team, “ORF3b and ORF8 are the least identical proteins to SARS-CoV-2, and they do not exist in other strains of human coronaviruses.”

The researchers say the performance of their test needs to be confirmed and refined using more significant numbers of COVID-19 patients and uninfected controls.

They also say that they still need to explore whether antigens aside from spike provide protection.

“Such information will help prioritize antigen targets for vaccine development, monoclonal antibody reagents and detecting early responses to infection,” conclude Valkenburg and the team.

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

Journal reference:
Valkenburg S, et al. Beyond the Spike: identification of viral targets of the antibody response to SARS-CoV-2 in COVID-19 patients. medRxiv 2020. https://www.medrxiv.org/content/10.1101/2020.04.30.20085670v1