Researchers in the United States have shown that an antibody test measuring host reactivity to the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may better detect natural infection among vaccinated individuals than an assay that measures reactivity to the viral spike protein.
The novel SARS-CoV-2 virus is the agent responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic that continues to pose a significant threat to global public health and the worldwide economy.
The viral spike protein mediates the initial stage of the infection process by binding to host cell receptors, while the nucleocapsid protein is involved in packaging the viral genome into new virions once SARS-CoV-2 has accessed a cell.
The spike protein is the primary target of neutralizing antibodies following natural infection or vaccination.
The currently approved vaccines that are being rolled out in many countries are based only on the spike protein. Antibody assays testing for reactivity to the spike, therefore, generate positive results following either natural infection or vaccination.
Now, a team from the University of Minnesota, Quansys Biosciences in Utah, the University of Chicago and the Minnesota Department of Health have conducted a study showing that in the context of vaccination, a nucleocapsid-containing assay was an accurate predictor of natural infection, while a spike-only assay performed poorly.
Ryan Demmer and colleagues say that, to their knowledge, this is the first investigation of this nature.
“In the era of SARS-CoV-2 vaccination, seroprevalence studies monitoring natural infection will require assays that do not rely on the spike-protein response alone,” writes the team.
A pre-print version of the research paper is available on the medRxiv server, while the article undergoes peer review.
Antibody assays are important for monitoring natural infection rates
Identifying cases of SAR-CoV-2 using antibody assays is important for monitoring rates of natural infection.
However, most currently authorized antibody tests cannot distinguish natural infection from vaccination; these assays detect reactivity to the viral spike protein, which triggers antibody responses in the case of both vaccination and infection.
“Consequently, ongoing antibody studies will be unable to accurately differentiate prior SARS-CoV-2 infection from vaccination against SARS-CoV-2 in populations with high vaccination coverage,” writes Demmer and the team.
On the other hand, the currently authorized vaccines that are being rolled out in many countries are not expected to elicit a nucleocapsid response.
Antibody tests that target both spike and nucleocapsid proteins could, therefore, potentially identify prior infection among vaccinated individuals.
What did the researchers do?
The team compared the accuracy of a nucleocapsid-containing assay with that of a spike-only assay in identifying prior SARS-CoV-2 infection among a cohort of healthcare workers (aged 18 to 80 years; median age 40 years) in the Minneapolis/St. Paul metropolitan area.
Two rounds of seroprevalence studies, separated by one month, were conducted between November 2020 and January 2021.
Blood samples collected during rounds 1 and 2 were tested for immunoglobulin G (IgG) antibodies reactive to the SARS-CoV-2 spike protein using a qualitative ELISA (spike-only assay).
In a subsample of participants (n=82) with enough blood available for further testing at round 2, a second assay was performed that measured IgGs against the SARS-CoV-2 nucleocapsid protein (nucleocapsid-containing assay).
All the round 1 blood samples were collected from participants before they had received a vaccine and vaccination status was determined via self-report at round 2.
What did the study find?
The seroprevalence at round 1 was 9.5% and 46% of participants reported being vaccinated prior to the round 2 testing.
Among those who reported being vaccinated, the mean time between vaccination and antibody testing at round 2 was nine days (range: 1 to 34 days).
Among individuals who had not recently been infected, the median value obtained from the spike-only assay was significantly elevated. In contrast, no significant difference was observed in the value obtained from the nucleocapsid-containing assay.
Across all participants, reactivity to the nucleocapsid protein accurately predicted recent infection.
For individuals who had been vaccinated more than ten days prior to the round 2 testing, the specificity of the nucleocapsid-containing assay was 92%, while the specificity of the spike-only assay was 0%.
Among participants with apparent false-positive results for the nucleocapsid-containing assay (i.e., a positive nucleocapsid result at round 2, but a negative spike result at round 1), the median time between the two sampling rounds was 33.5 days.
Demmer and colleagues say this raises the possibility that these individuals were “truly infected in the interim.”
What did the authors conclude?
“Among vaccinated individuals, the nucleocapsid assay had a substantially higher specificity and positive predictive value, compared to the spike-only assay,” say the researchers.
“These findings suggest that in the era of SARS-CoV-2 vaccination, seroprevalence studies monitoring natural infection will require assays that capture reactivity to the nucleocapsid protein,” concludes the team.
medRxiv 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.