The current worldwide outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has wreaked devastating economic and social impacts, besides the staggering toll on human life. To rapidly diagnose and treat or isolate COVID-19-positive cases, there is a need to have a reliable bedside test for this virus.
Study: Finger stick blood test to assess post vaccination SARS-CoV-2 neutralizing antibody response against variants. Image Credit: rootstudio/ Shutterstock
A new preprint reports a semi-quantitative test that can measure neutralizing antibodies to the SARS-CoV-2 pathogen at the point of care (POC), using a finger stick or venipuncture blood. The test will be able to evaluate serologic responses against the wild-type virus, as well as against the newer variants, the Alpha, Beta, Gamma, and Delta variants.
The study demonstrates good concordance with earlier results while also showing a significant reduction in neutralizing titer for the Beta, Gamma, and Delta variants in the group who had been vaccinated with the Pfizer/BioNTech messenger ribonucleic acid (mRNA) vaccines more than three months before testing, compared to those who had received it within the last three months.
The virus has mutated, and new variants of concern (VOCs) have emerged, as named above, with much higher transmissibility and immune evasion tactics, allowing them to spread more rapidly and cause more severe disease as well as breakthrough infections in the vaccinated population. These have become dominant in successive waves, with the Delta variant causing the overwhelming majority of infections at present.
Both virus neutralization tests and pseudovirus neutralization tests (VNT and pVNT, respectively) are used to assess protective immune responses. Still, they can be carried out only in biosafety laboratories level 2 or 3 as requiring skill and much time. Many variables affect their outcome, and hence their reproducibility is often limited.
An acceptable substitute is the neutralization test based on the enzyme-linked immunosorbent assay (ELISA), but this also depends on operator skill and sophisticated facilities. The currently available POC tests are designed to detect total antibody (immunoglobulin) levels, thus providing an unreliable correlate of immune protection or a qualitative picture only.
The current study, on the medRxiv* preprint server, aims to help fulfill this perceived need by describing an accurate and rapid POC test that can measure the immune antibody response to vaccination, especially the protection offered against infection with VOCs.
An earlier paper by the same authors presented a paper-based SARS-CoV-2 neutralization assay (the cellulose pulled-down virus neutralization test (cpVNT)), capable of identifying neutralizing antibodies to the virus within 10 minutes, though it could be used only with serum or plasma. The test detected the viral receptor-binding domain (RBD) in complex with the angiotensin-converting enzyme 2 (ACE2) receptor for the virus on the host cell. Its disruption by the presence of neutralizing antibodies, which would reduce the overall signal.
This test was adapted for use on whole blood, making it more suitable for POC testing. Not only did it show good agreement with the pVNT and ELISA-based assays. The test can be used to assess the neutralizing responses to the SARS-CoV-2 VOCs in the vaccinated population as well.
The key changes made included shifting to the use of a fluorescent reporter molecule and sequential incubation steps. This allowed for better neutralization of the RBD-ACE2 binding.
What did the study show?
The results showed that the neutralizing activity was different in the pre- and 1-2, and 3-4 weeks post-first dose vaccination samples with both types of vaccines, the Pfizer/BioNTech and Moderna vaccines. The values were ~2% vs. 14% and ~37%, respectively. After the second dose, the median blocking was 89%.
At the post-vaccination sample, the samples showed different median blocking activity between the two vaccines, while the later samples were more comparable in their inhibitory activity. The modified test could detect neutralizing activity over a wider range than either sVNT or pVNT, thus ensuring its clinical applicability within 10 minutes.
The percentage of inhibition in blood samples is about 0.87 times that observed in plasma samples. The median percentage of blocking in the pre-vaccination samples was less than a third of that in the current test.
The test was also more than 80% sensitive and 100% specific compared to commercial tests, with lower specificity and sensitivity than the ELISA and pVNT, probably due to the different types of samples and assays.
The types of blood samples, venous vs. finger stick, also showed excellent correlation between themselves when the pre-vaccination, early vaccinated, and later vaccinated samples were compared with this test, showing its potential utility for finger stick sampling.
The study also showed that the mutations in the RBD in the various VOCs increased the binding affinity compared to the wild-type, especially the Gamma, which showed a 3-fold rise compared to the ancestral variant.
They examined a hypothetical variant that contained both the characteristic N501Y and the T478K mutations. Indeed, it had the highest binding affinity for ACE2. All VOCs had resulting high ACE2 affinity compared to the wild-type virus; the variant described here did not show above 1.6-fold 9
The nAb percent blocking against Beta and Gamma strains were markedly decreased, unlike the other two VOCs, while with the engineered variant RBD, the vaccine-elicited neutralizing antibody could displace ACE2 from the RBD, being reduced significantly to 72.4 %. Neutralizing responses were most significantly affected for these strains in people who had received the Pfizer vaccine.
Over time, neutralizing activity dropped with the wild-type and Alpha RBD binding, but more significantly with the Beta, Gamma, and Delta variants, in contrast to the stability of neutralizing responses to the wild-type virus. This corroborates recent studies of waning protection following the Pfizer vaccine, indicates the utility of the test to evaluate vaccine-induced neutralizing immunity, and identifies those who would benefit from a third booster dose.
What are the implications?
The researchers point to this test as a tool to better analyze the neutralizing response to the virus vs. the extent of RBD-ACE2 binding, more particularly when it comes to newer variants. The hypothetical variant described above showed no immune evasion capability, with over 90% blocking by neutralizing antibodies even though it had a high ACE2 binding affinity. These mutations are therefore of no importance in neutralizing antibody binding.
The Beta had only slightly greater binding affinity but much higher immune evasion, with much lower percent blocking of neutralizing antibodies compared to the wild-type virus. The potent combination of RBD-ACE2 binding affinity with low neutralizing antibody recognition means a much greater immune escape.
Cellular immunity is, of course, the key to providing durable immunity against the disease, but this test allows reliable detection of immune protection at POC level. About 90% of neutralizing activity in convalescent plasma is directed at the immunodominant RBD 25-27, which makes this test, with its standardized percent blocking measurement, a consistent one.
Moreover, it is simple to adapt to the identification of a new variant, requiring only a change in a single reagent, allowing scientists to keep up with the virus as it rapidly changes and evolves. Its ability to detect waning immunity as a POC test is also important, agreeing with the increased breakthrough infections among those who were vaccinated with the Pfizer vaccine from January to April 2021.
This test is highly suited for PoC settings and provides an insightful nAb response in a post-vaccinated population. [This] would be a valuable tool in enabling public health authorities to manage breakthrough infections and to develop an effective booster vaccination strategy for more vulnerable individuals.”
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.