In a recent study posted to the medRxiv preprint* server, researchers pursued evidence of whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens, of which spike (S) protein is highly immunogenic, persist beyond the acute phase of coronavirus disease 2019 (COVID-19).

Study: Plasma-based antigen persistence in the post-acute phase of SARS-CoV-2 infection. ​​​​​​​Image Credit: Created with the assistance of DALL·E 3

*Important notice: medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Background

Infections caused by most ribonucleic acid (RNA) viruses are transient. Thus, researchers postulated that COVID-19, caused by SARS-CoV-2, an RNA virus, is not a persistent infection; however, evidence points to a different reality.

Studies suggest that SARS-CoV-2 antigens, particularly spike subunits 1 and 2 (S1 and S2) and nucleocapsid (NC), might persist following COVID-19. Studies also indicate that several virologic and immunologic factors work behind the persistence of SARS-CoV-2 antigens beyond the acute phase of illness.

However, the results of these studies are limited, given the need for a large sample set of negative control samples. 

About the study

In the present study, researchers developed two cohorts comprising participants from the prepandemic (before 2019) and pandemic era to test their plasma samples for the presence of S, S1, and NC antigens using single molecule array (SMA) assays. 

The pre-pandemic plasma samples were collected randomly from the participants of the Study of the Consequences of the Protease Era (SCOPE) cohort study done at the University of California, San Francisco (UCSF) in 2001 to investigate the pathogenesis of human immunodeficiency virus (HIV). For each HIV-infected participant, four uninfected participants of matching age and race/ethnicity provided samples for the current analysis.

They collected stored plasma samples from the participants of the Long-term Impact of Infection with Novel Coronavirus (LIINC) study completed at UCSF, who made the most study visits in the 1.25 years following the COVID-19 pandemic. 

Further, the researchers administered questionnaires in both groups to collect data on sociodemographic, economic, and clinical characteristics. In the pandemic-era group, they specifically inquired about self-reported symptoms experienced and perception of overall health on a scale of zero to 100 in the first three weeks of the acute phase of COVID-19 and whether COVID-19-related hospitalization occurred. Additionally, they monitored SARS-CoV-2 vaccinations and any additional SARS-CoV-2 infections since the index infection.

The S, S1, and NC assays were performed separately, using antibodies specific to S1, S2, and NC attached to carboxylated magnetic beads. The team calculated average enzyme per bead (AEB) values and separately reported values above the limit of detection (LOD) as the background AEB. Further, they used a four-parameter logistic regression and prevalence ratios (PRs) to depict the relationship of demographic and clinical factors with antigen persistence across both study groups. 

In statistical analysis, assays evaluating pandemic samples monitored antigen presence during 3, 6, to 10 months and 11 to 14 months post-onset of COVID-19 symptoms. They defined the presence or absence of each antigen in all three assays and at least one antigen versus all three antigens.

Results

The pandemic- and pre-pandemic groups were similar in attributes; however, they were variable in the proportion of women. The former had 50% women, with an average age of 46 years, while the latter comprised only 22% women. The study follow-up for pandemic-era participants continued long after index infection, between 0.9 and 15.4 months. 

SARS-CoV-2 antigens, S, S1, and NC were present in three, three, and two samples of the total 250 pre-pandemic samples, respectively. The presence of at least one antigen in all 250 plasma samples suggested the prevalence of false positivity. 

The presence of any SARS-CoV-2 antigen was relatively more frequent in all pandemic-era time points, driven by NC in the first six and S for up to 14 months after index SARS-CoV-2 infection. Notably, a single antigen was detected in most pandemic-era cases (59/61, 96.7%).

The detection of multiple antigens at a single time point was uncommon. Accordingly, all three antigens were not detected simultaneously; however, two antigens (one for S1 and N each) were simultaneously detected. In samples with detectable antigens, the average S, S1, and N concentrations were 27.7 pg/mL, 31.2 pg/mL, and 23.6 pg/mL, respectively.

Among vaccinated individuals, antigens were detected within three weeks of receiving a vaccine dose in five instances (three for S1 and one each for S and N).

Among other determinants of antigen positivity among pandemic-era participants, evidence of an association between age, gender, race/ethnicity, body mass index (BMI), or HIV status was lacking. However, the influence of the severity of the acute phase of illness was strong.

Thus, participants who required hospitalization for acute COVID-19 were two times more likely to have an antigen detected (PR=1.86, p=0.03), while those not hospitalized for COVID-19 but having severe acute illness were over three times more likely to have antigens detected than those with less severe acute illness (PR=3.5, p=0.07).

Conclusions

Studies have confirmed a link between persistence of SARS-CoV-2's antigens and post-acute sequelae of SAR-CoV-2 (PASC). Accordingly, even in this study, the researchers detected potentially immunogenic viral antigens in over 10% of plasma samples from COVID-19 patients during the pandemic era for up to 14 months following index SARS-CoV-2 infection, providing robust evidence that SARS-CoV-2 is a unique RNA virus. 

Given the millions of people SARS-CoV-2 has infected, and its S is a highly immunogenic antigen, this phenomenon has significant implications. First and foremost, it implies that, unlike most other RNA viruses, SARS-CoV-2 infection is not transient in all cases, and it can evolve in immunocompromised individuals.

However, unlike previous studies, which examined SARS-CoV-2 antigens' persistence in patients seeking clinical care in PASC clinics, researchers of this study used samples from individuals in the post-acute phase of COVID-19 regardless of their symptom severity. Moreover, they carried out a direct comparison of SARS-CoV-2 antigen persistence in the post-acute phase between pre-pandemic (control) and pandemic samples, which mitigated all concerns of attaining false positive results. 

Further work is warranted to assess whether the persistence of SARS-CoV-2 antigens is a driver of PASC or simply an insignificant consequence of previous infection. Furthermore, studies with larger sample sizes are needed to determine whether antigen persistence is causally related to symptoms.

*Important notice: 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.

Written by

Neha Mathur

Neha is a digital marketing professional based in Gurugram, India. She has a Master’s degree from the University of Rajasthan with a specialization in Biotechnology in 2008. She has experience in pre-clinical research as part of her research project in The Department of Toxicology at the prestigious Central Drug Research Institute (CDRI), Lucknow, India. She also holds a certification in C++ programming.