Natural infection or vaccination can both provide humoral immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The majority of patients infected with coronavirus disease 2019 (COVID-19) exhibited long-term serological responses. Although antibodies to SARS-CoV-2 can be found in the majority of SARS-CoV-2 infected people, antibody levels are widely diverse.

Study: mRNA vaccine-induced antibodies more effective than natural immunity in neutralizing SARS-CoV-2 and its high affinity variants. Image Credit: Studio Romantic/Shutterstock

Many of the current methods, on the other hand, have difficulties in detecting circulating antibodies or neutralizing activity in those patients. In one case, those with semi-quantitative IgG antibody findings and titers of less than 1:320 were unable to achieve the appreciable neutralizing activity.

The SARS-CoV-2 spike (S) protein attaches to angiotensin-converting enzyme 2 (ACE2) via its receptor-binding domain (RBD), which is becoming a significant public health study subject because of its involvement in producing neutralizing antibodies and mutations in emerging SARS-CoV-2 variants, that are spreading fast around the world.

Antisera efficiently neutralized the first of these variations, which had a D614G mutation in the spike protein and increased viral titer and infectivity. A UK variant B.1.1.7 was recently linked to an increase in COVID-19 patients. It contained the N501Y mutation in the RBD area, which is important in contacting ACE2. N501Y, as well as two other RBD domain alterations, K417N/T and E484K, were later discovered in SARS-CoV-2 strains from South Africa (B.1.351)25 and Brazil (P.1).

Antibody quantification and functional neutralization assays were created in this study by a group of researchers from several institutions. RBD antibody levels are an effective proxy for biochemical neutralizing activities, according to analyses of both COVID-19 convalescent and diagnostic cohorts. The mRNA samples obtained from vaccinated patients also showed a median of 17 times greater RBD antibody levels and a similar level of elevated neutralization activity against RBD-ACE2 binding as those from infections that occurred naturally, according to the findings.

The study

The authors designed an electrochemiluminescence-based serology test with remarkable sensitivity to precisely quantify the amounts of anti-RBD antibodies in individuals infected with SARS-CoV-2. For optimum selectivity, the test relies on two antibody-antigen interactions. The researchers isolated recombinant wildtype (WT) RBD from transiently transfected 293 cells and marked it with biotin or ruthenium (Ru)-tag for antibody capture or detection.

The test was clinically validated using 41 specimens from 33 previously infected donors with a known history of COVID-19 collected before the first documented alpha variant containing the RBD mutation N501Y, and 171 healthy donors obtained prior to January 2020. COVID vaccinations were not given to any of the previously infected donors.

All negative specimens were found to be less than the lower limit of quantification (LLoQ), although all previously infected donors tested positive. Throughout this small cohort of convalescent samples, the median RBD antibody level was 1.33 g/mL, with a significant 170-fold range for RBD antibody levels. With this group of 41 specimens from those with previously confirmed COVID-19 infections, the RBD antibody assay had an area under curve (AUC) of 1.00 in Receiver Operator Characteristic (ROC) analysis, giving it 100% sensitivity. It also has 100% specificity because of the lack of detectable antibody levels in pre-COVID-19 donors' serum samples.

The scientists devised an electrochemiluminescence-based protein binding test, utilizing recombinant RBD and ACE2 proteins, to properly assess the antisera's ability to neutralize RBD and ACE2 binding. The 41 samples of COVID-19 sera had a considerably greater inhibitory impact against RBD-ACE2 binding, according to the findings.

The sera obtained from previously infected individuals had a median inhibition level of 93%, while the control sera had a median inhibition level of 7%. The antibody neutralization assay had high sensitivity and specificity when compared to the negative controls, with an AUC in ROC analysis of 0.986, indicating good sensitivity and specificity of the assay.

The authors isolated N501Y RBD protein and labeled it with the Ru-tag for electro-chemiluminescence assay to see if antisera from previously infected patients could recognize the B.1.1.7 N501Y variant (alpha). When the antisera from individuals previously infected with COVID-19 were evaluated against the pre-COVID-19 donor sera specimens, the results demonstrated that the antisera from those previously infected with COVID-19 neutralized the antisera specifically. With an AUC of 0.948, the neutralization assay against RBD-N501Y and ACE2 binding was both sensitive and specific. Furthermore, with a slope of 1.03, there was a substantial linear association between neutralizing activity against the WT and the N501Y RBD in ACE2 binding. Therefore, equal neutralization from COVID-19 antisera is seen in both WT and N501Y RBD.

Implications

When compared to those who became seropositive owing to previous COVID-19 infection, the investigators found that blood from mRNA vaccinated individuals’ blood has a median of 17 times greater RBD antibody levels. The ability to biochemically neutralize RBD binding to the cellular ACE2 receptor was found to be highly correlated with high RBD antibody levels in these findings.

Written by

Colin Lightfoot

Colin graduated from the University of Chester with a B.Sc. in Biomedical Science in 2020. Since completing his undergraduate degree, he worked for NHS England as an Associate Practitioner, responsible for testing inpatients for COVID-19 on admission.