Abstract
The SARS-CoV-2 pandemic has spread to all parts of the world and can cause life-threatening pneumonia and other severe disease manifestations known as COVID-19. This health crisis has resulted in a significant effort to stop the spread of this new coronavirus. However, while propagating itself in the human population, the virus accumulates mutations and generates new variants with increased fitness and the ability to escape the human immune response. Here we describe a color-based barcoded spike flow cytometric assay (BSFA) that is particularly useful to evaluate and directly compare the humoral immune response directed against either wild type (WT) or mutant spike (S) proteins or the receptor-binding domains (RBD) of SARS-CoV-2. This assay employs the human B lymphoma cell line Ramos, transfected for stable expression of WT or mutant S proteins or a chimeric RBD-CD8 fusion protein. We find that the alpha and beta mutants are more stably expressed than the WT S protein on the Ramos B cell surface and/or bind with higher affinity to the viral entry receptor ACE2. However, we find a reduce expression of the chimeric RBD-CD8 carrying the point mutation N501Y and E484K characteristic for the alpha and beta variant, respectively. The comparison of the humoral immune response of 12 vaccinated probands with 12 COVID-19 patients shows that after the boost, the S-specific IgG class immune response in the vaccinated group is similar to that of the patient group. However, in comparison to WT the specific IgG serum antibodies bind less well to the alpha variant and only poorly to the beta variant S protein. This is in line with the notion that the beta variant is an immune escape variant of SARS-CoV-2. The IgA class immune response was more variable than the IgG response and higher in the COVID-19 patients than in the vaccinated group. In summary, we think that our BSFA represents a useful tool to evaluate the humoral immunity against emerging variants of SARS-CoV-2 and to analyze new vaccination protocols against these variants.
Highlights
Since December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has been spreading in the human population as a pathogen causing the coronavirus disease 2019 (COVID-19) associated with severe pneumonia [1]
The Ramos-null cells carry on their surface a receptor for ecotropic retroviruses that allows the efficient transduction of these cells with murine retroviral vectors (Figure 1A)
For the expression of different S constructs on the surface of Ramos-null cells, we used a pMIG vector carrying after the LTR promoter the construct sequence, an internal ribosome entry site (IRES) and the sequence coding for the green fluorescent protein (GFP) (Figure 1B)
Summary
Since December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has been spreading in the human population as a pathogen causing the coronavirus disease 2019 (COVID-19) associated with severe pneumonia [1]. SARS-CoV-2 enters the cells via the angiotensin-converting enzyme 2 (ACE2), a receptor widely expressed in human mucosal tissues of the nose and mouth and abundant in the lung [4, 5]. The virus binds to the ACE2 entry receptor via the trimeric spike (S) protein prominently expressed on the viral membrane [7]. Upon binding to ACE2, the S protein undergoes a conformational change. It is cleaved by cellular proteases into an S1 and S2 portion, with the latter inducing a fusion reaction between the viral and cellular membrane, thereby starting the infection cycle [8]. It has revealed that the RBD can assume a closed (down) or open (up) conformation, with only the latter being able to interact with the ACE2 entry receptor [9]
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