Comprehensive analysis of immune responses to SARS-CoV-2

COVID-19 vaccination is one of the most important weapons to combat the pandemic. The main target used in COVID-19 vaccines is the viral spike protein. The receptor binding domain (RBD) of the S1 subunit of the spike protein is responsible for binding to the human cellular receptor angiotensin-converting enzyme 2 (ACE2) and mediating entry of the virus into the host cells.

Antibodies of class IgG against the S1/RBD and specific long-lived T cells appear to play the most important role in virus neutralization and sustained immunity. A trio of tests from EUROIMMUN for quantitative measurement of anti-S1/RBD IgG antibodies, determination of the neutralizing effect of these antibodies, and analysis of SARS-CoV-2-reactive T cells provides an all-round analysis of immune responses to SARS-CoV-2.

Antibody quantification

Quantitative determination of antibodies against S1/RBD is an important tool for assessing the individual immune response to SARS-CoV-2 after infection and measuring the immune reaction following vaccination with spike protein-based vaccines. Anti-SARS-CoV-2 S1/RBD IgG concentrations can be measured in serum, plasma, or dried capillary blood samples using the Anti-SARS-CoV-2 QuantiVac ELISA (IgG) from EUROIMMUN. The measurement is based on a six-point calibration curve, and the antibody concentrations are expressed in standardized binding antibody units (BAU/ml) according to the international reference material (NIBSC code: 20/136).

In a first study using the Anti-SARS-CoV-2 QuantiVac ELISA (IgG), the immune response was analyzed in 57 persons over a time frame of three weeks after first and second immunization with vaccines from Moderna, Pfizer/BioNTech, or AstraZeneca. The results showed that the longer the time interval following vaccination, the higher the antibody concentration was. Furthermore, different antibody concentrations were determined depending on the vaccine used. All samples taken after the second vaccination showed very high antibody concentrations.

Detection of neutralizing antibodies

Measurement of the neutralizing activity of anti-S1/RBD antibodies enables confirmation of their functionality in inhibiting binding of S1/RBD to ACE2 and thus preventing infection. The gold standard for determining neutralizing antibodies is the plaque reduction neutralization test (PRNT) or virus neutralization test (V-NT). These tests are, however, expensive and time-consuming to perform, and require biosafety level three laboratory conditions.

Surrogate virus neutralization tests offer a fast and economical alternative to PRNT. The SARS-CoV-2 NeutraLISA from EUROIMMUN detects anti-S/RBD antibodies that are capable of inhibiting binding of the RBD to ACE2. The assay is based on competitive binding between neutralizing antibodies in the patient samples and labeled ACE2 receptors to recombinant S1 coated onto microplate wells. The test demonstrates a very good (98.6%) agreement with the PRNT. It is, moreover, suitable for routine diagnostics, including in a high-throughput setting. The assay takes just 2 hours and is automatable.

The 57 samples described above were analyzed using the SARS-CoV-2 NeutraLISA. The results confirmed that the antibodies detected in the samples of the vaccinated study participants were for the most part neutralizing antibodies. These antibodies provide the greatest protection against SARS-CoV-2 infection.

Analysis of specific T cells

T-cell immunity, in particular against the spike protein, is associated with strong protection against SARS-CoV-2 and plays a particularly important role in patients who do not exhibit measurable concentrations of specific antibodies.

The T-cell-mediated cellular immune response to SARS-CoV-2 can be determined using the interferon gamma release assay (IGRA). The EUROIMMUN Quan-T-Cell ELISA used in combination with the Quan-T-Cell SARS-CoV-2 enables fast, automatable, and quantitative determination of the IFN-γ released by SARS-CoV-2-specific T cells. The T cells in the samples are stimulated using spike protein-based antigens in the provided tubes and the released IFN-γ is subsequently measured using a fully automated quantitative ELISA. The test is performed on heparinized whole blood samples, circumventing the need to prepare purified peripheral mononuclear cells (PBMCs). EUROIMMUN recommends a cut-off of 200 mIU/ml based on a study using 114 samples from healthy blood donors that were negative for both IgG and IgA antibodies against SARS-CoV-2.

In a further internal study, samples from 46 persons with a concordant positive or negative anti-SARS-CoV-2 antibody test result for IgG and IgA were investigated using the EUROIMMUN IGRA. There was a 93.8% agreement for positive samples and a 96.7% agreement for negative samples, demonstrating the concurrent presence of humoral and cellular responses in the majority of the tested persons.

The EUROIMMUN IGRA is well established in the research field and its high quality has been proven in many external studies (e.g. 1, 2, 3, 4). The diagnostic sensitivity based on a study using 143 samples from convalescent and vaccinated individuals amounted to 97.9%, while the diagnostic specificity in 45 samples from unvaccinated individuals without a COVID-19 infection history was 97.8% (data from ref. 1 adapted to EUROIMMUN’s cut-off recommendations).

The IGRA has also been used, alongside antibody testing, to investigate immune responses after vaccination in vulnerable groups such as immunocompromised individuals, elderly persons, or dialysis patients (1, 2, 4). Since at-risk persons may exhibit reduced or inadequate immune responses, evaluation of both arms of the immune response is critical, especially when considering potential strategies for booster shots.

Perspectives

A broad analysis of both humoral and cellular immune responses to SARS-CoV-2 is critical for evaluating the efficacy of vaccines. A thorough understanding of immune reactions will enable optimization of vaccine strategies with regard to dosage, time intervals, boosters, and individual circumstances such as previous infection or a compromised immune system. Examination of immune responses is also important for establishing the durability of immunological memory and cross protection between SARS-CoV-2 variants. Furthermore, antibody and T-cell immunity analyses aid epidemiological surveillance of the spread of COVID-19 and the extent of population-level immunity.

References

Huzly et al. Validation and performance evaluation of a novel interferon-γ release assay for the detection of SARS-CoV-2 specific T-cell response. medRxiv 2021.07.17.21260316 (2021).

Schwarz et al. Delayed Antibody and T-Cell Response to BNT162b2 Vaccination in the Elderly, Germany. Emerg Infect Dis 27(8):2174-2178 (2021).

Hillus et al. Safety, reactogenicity, and immunogenicity of homologous and heterologous prime-boost immunization with ChAdOx1-nCoV19 and BNT162b2: a prospective cohort study. Lancet Respir Med (2021) Online ahead of print

Schrezenmeier et al. Immunogenicity of COVID-19 Tozinameran Vaccination in Patients on Chronic Dialysis. Front. Immunol. 12:690698 (2021)