Analysis of SARS-CoV-2 specific humoral and cellular immune reactions is critical for assessing the efficacy of COVID-19 vaccines.

Most currently used vaccines are based on the SARS-CoV-2 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 neutralisation and sustained immunity. 

EUROIMMUN offers a powerful trio of CE-marked test systems for quantitative measurement of anti-S1/RBD IgG antibodies, determination of the neutralising effect of these antibodies, and analysis of SARS-CoV-2-reactive T cells:

• Anti-SARS-CoV-2 QuantiVac ELISA (IgG): CE-marked ELISA for quantification of the anti-S1/RBD IgG concentration in standardized units (BAU/ml)
• SARS-CoV-2 NeutraLISA: CE-marked surrogate virus neutralisation test in ELISA format for detection of neutralising antibodies targeting S1/RBD
• Quan-T-Cell SARS-CoV-2 / Quan-T-Cell ELISA: CE-marked interferon-gamma release assay (IGRA) for quantitative determination of the IFN-γ released by SARS-CoV-2-specific T-cells 

This all-around analysis supports assessment of immune responses following infection or vaccination with spike protein-based vaccines.

Quantitative antibody detection
Quantitative determination of antibodies against S1/RBD aids assessment of the individual immune response to SARS-CoV-2 after infection and measurement of the immune reaction after vaccination.

The Anti-SARS-CoV-2 QuantiVac ELISA (IgG) is a CE-marked and fully automatable assay for quantification of the anti-S1/RBD IgG antibody concentration in standardized binding antibody units (BAU/ml) according to international reference material (NIBSC code: 20/136) using a six-point calibration curve.

The assay shows an excellent correlation with the international standard (Figure 1). The Anti-SARS-CoV-2 QuantiVac ELISA (IgG) also shows a very good result agreement with different neutralisation tests.

The test is validated for serum, plasma, and dried capillary blood as sample material. 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 second vaccination showed very high antibody concentrations.

Figure 1. Correlation of the Anti-SARS-CoV-2 QuantiVac ELISA (IgG) with the WHO international standard

Determination of neutralising antibodies
Measurement of the neutralising activity of anti-S1/RBD antibodies enables confirmation of their functionality in inhibiting binding of S1/RBD to ACE2 and preventing infection of the host cells.

The CE-marked SARS-CoV-2 NeutraLISA is a surrogate virus neutralisation test (sVNT) in ELISA format for determination of the inhibiting effect of anti-SARS-CoV-2 antibodies.

The test procedure is based on competitive binding between neutralising antibodies in the patient samples and biotinylated ACE2 receptors to recombinant S1 coated onto microplate wells. The higher the concentration of neutralising antibodies in the sample is, the weaker the color reaction will be.

The assay offers a fast and economical alternative to the gold standard PRNT, which is expensive and time-consuming to perform. In contrast to the PRNT, the SARS-CoV-2 NeutraLISA does not require the use of biosafety level three (BSL-3) laboratory conditions.

It takes just 2 hours to perform and is automatable, making it suitable for routine diagnostics, including at high throughput. The test demonstrates a 98.6% agreement of qualitative results with the PRNT (Table 1).

In the study described above, the 57 samples were additionally 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 neutralising antibodies. These antibodies provide the greatest protection against SARS-CoV-2 infection. 

Table 1. Comparison of results from SARS-CoV-2 NeutraLISA and PRNT₅₀

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 CE-marked 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.

In the test procedure, the T cells in the patient 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 assay 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.

Conclusions
The test trio from EUROIMMUN provides a comprehensive analysis of humoral and cellular immune responses to SARS-CoV-2, supporting evaluation of the efficacy of vaccines as well as epidemiological studies on the spread of COVID-19.

Further information at www.coronavirus-diagnostics.com

References
1. 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).

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

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

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