Rapid finger test for antibodies to SARS-CoV-2 variants

The current global coronavirus disease 2019 (COVID-19) epidemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has had devastating economic and social impacts, in addition to the staggering toll of lives human. To quickly diagnose and treat or isolate positive cases for COVID-19, it is necessary to have a reliable bedside test for this virus.

To study: Finger blood test to assess the response of neutralizing antibodies to SARS-CoV-2 after vaccination against variants. Image Credit: rootstudio / Shutterstock

New preprint reports semi-quantitative test that can measure neutralizing antibodies the SARS-CoV-2 pathogen at the point of care (POC), using a finger prick or blood drawn by venipuncture. The test will be able to assess serological responses against the wild-type virus, as well as against the newer variants, Alpha, Beta, Gamma and Delta variants.

The study demonstrates good agreement with previous results while also showing a significant reduction in neutralizing titer for beta, gamma and delta variants in the group that had been vaccinated with Pfizer / BioNTech plus messenger ribonucleic acid (mRNA) vaccines. three months ago. test, compared to those who had received it in the past three months.

Background

The virus mutated and new variants of concern (VOCs) appeared, as noted above, with much higher transmissibility and immune evasion tactics, allowing them to spread faster and cause more serious illness as well. as revolutionary infections in the vaccinated population. These have become dominant in successive waves, with the Delta variant causing the overwhelming majority of infections today.

Virus neutralization tests and pseudovirus Neutralization tests (VNT and pVNT, respectively) are used to assess protective immune responses. However, they can only be carried out in biosafety level 2 or 3 laboratories because they require skills and a lot of time. Many variables affect their result and therefore their reproducibility is often limited.

An acceptable substitute is the enzyme-linked immunosorbent assay (ELISA) -based neutralization test, but this also depends on operator skills and sophisticated facilities. Currently available POC tests are designed to detect levels of total antibodies (immunoglobulins), thus providing an unreliable correlate of immune protection or a qualitative picture only.

The study in progress, on the medRxiv* preprint server, aims to help meet this perceived need by describing an accurate and rapid POC test that can measure the immune response of antibodies to vaccination, in particular the protection offered against VOC infection.

An earlier article by the same authors presented a paper-based SARS-CoV-2 neutralization test (the Cellulose Extraction Virus Neutralization Test (cpVNT)), capable of identifying neutralizing antibodies against the virus within 10 minutes. , although it can only be used 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 disturbance by the presence of neutralizing antibodies, which would reduce the overall signal.

This test has been adapted for use on whole blood, which makes it more suitable for POC testing. Not only did it show good agreement with the pVNT and ELISA tests. The test can also be used to assess neutralizing responses to SARS-CoV-2 VOCs in the vaccinated population.

The main changes included the switch to the use of a fluorescent reporter molecule and sequential incubation steps. This allowed better neutralization of the RBD-ACE2 binding.

What did the study show?

The results showed that the neutralizing activity was different in the vaccination samples before, 1-2 and 3-4 weeks after the first dose with the two types of vaccines, the Pfizer / BioNTech and Moderna vaccines. The values ​​were about 2% versus 14% and about 37%, respectively. After the second dose, the median blockade was 89%.

In the post-vaccination sample, the samples showed a 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 sVNT or pVNT, thus ensuring its clinical applicability within 10 minutes.

The percent inhibition in blood samples is approximately 0.87 times that seen in plasma samples. The median percent blockage in the pre-vaccination samples was less than a third of that in the current test.

The test was also over 80% sensitive and 100% specific compared to commercial tests, with lower specificity and sensitivity than ELISA and pVNT, possibly due to the different types of samples and tests.

Types of blood, venous vs. finger prick, also showed excellent correlation between them when pre-vaccine, early vaccinated and later vaccinated samples were compared to this test, showing its potential utility for finger prick sampling.

The study also showed that RBD mutations in the various VOCs increased binding affinity compared to wild type, in particular Gamma, which showed a 3-fold increase compared to the ancestral variant.

They examined a hypothetical variant that contained both the characteristic N501Y and T478K mutations. Indeed, it had the highest binding affinity for ACE2. All VOCs exhibited a high affinity for ACE2 compared to the wild type virus; the variant described here did not show more than 1.6 times 9

The percentage of blockade of nAb against the beta and gamma strains was markedly decreased, unlike the other two VOCs, while with the modified RBD variant, the neutralizing antibody elicited by the vaccine could displace ACE2 from RBD, being reduced. significantly at 72.4%. Neutralizing responses were more significantly affected for these strains in people who received the Pfizer vaccine.

Over time, neutralizing activity decreased with wild-type and Alpha RBD binding, but more significantly with beta, gamma, and delta variants, in contrast to the stability of neutralizing responses to wild-type virus. This corroborates recent studies on decreased protection after Pfizer vaccine, indicates the test’s usefulness in assessing vaccine-induced neutralizing immunity, and identifies those who would benefit from a third booster dose.

What are the implications?

Researchers see this test as a tool to better analyze the neutralizing response to the virus with respect to the extent of RBD-ACE2 binding, especially when it comes to newer variants. The hypothetical variant described above showed no immune evasion ability, with over 90% blockage by neutralizing antibodies, even though it had high ACE2 binding affinity. These mutations are therefore of no importance in the neutralization of antibody binding.

Beta had only slightly higher binding affinity but much higher immune evasion, with a much lower percentage of neutralizing antibody blockade compared to wild-type virus. The potent combination of RBD-ACE2 binding affinity with poor recognition of neutralizing antibodies means much greater immune evasion.

Cellular immunity is, of course, the key to providing long-lasting immunity against disease, but this test allows reliable detection of immune protection at the POC level. About 90% of the neutralizing activity in convalescent plasma is directed against the immunodominant RBD 25-27, making this assay, with its standardized percent blocking measure, a consistent assay.

In addition, it is simple to adapt to the identification of a new variant, requiring only a change of a single reagent, allowing scientists to follow the virus as it changes and evolves. quickly. Its ability to detect decline in immunity as a POC test is also important, in line with the increase in breakthrough infections among those who were vaccinated with the Pfizer vaccine from January to April 2021.

This assay is ideally suited to the PoC parameters and provides an insightful nAb response in a post-vaccinated population. [This] would be a valuable tool to enable public health authorities to manage breakthrough infections and develop an effective booster vaccination strategy for the most vulnerable people. “

*Important Notice

medRxiv publishes preliminary scientific reports which are not peer reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behavior, or treated as established information.