Antibody Levels Poorly Reflect on the Frequency of Memory B Cells Generated following SARS-CoV-2, Seasonal Influenza, or EBV Infection

Abstract

The scope of immune monitoring is to define the existence, magnitude, and quality of immune mechanisms operational in a host. In clinical trials and praxis, the assessment of humoral immunity is commonly confined to measurements of serum antibody reactivity without accounting for the memory B cell potential. Relying on fundamentally different mechanisms, however, passive immunity conveyed by pre-existing antibodies needs to be distinguished from active B cell memory. Here, we tested whether, in healthy human individuals, the antibody titers to SARS-CoV-2, seasonal influenza, or Epstein–Barr virus antigens correlated with the frequency of recirculating memory B cells reactive with the respective antigens. Weak correlations were found. The data suggest that the assessment of humoral immunity by measurement of antibody levels does not reflect on memory B cell frequencies and thus an individual’s potential to engage in an anamnestic antibody response against the same or an antigenically related virus. Direct monitoring of the antigen-reactive memory B cell compartment is both required and feasible towards that goal.

Four-color ImmunoSpot® assays requiring only 1-3 mL of blood permit precise frequency measurements of antigen-specific B cells secreting immunoglobulins of all four classes and subclasses

“…If a special protocol is followed, PBMC can be frozen without impairing the B cells' functionality ( [7], and N. Becza manuscript in preparation). Thus, by freezing B cells of a sample in several aliquots, the same PBMC can be tested repeatedly, reproducing the results of the previous experiment with high accuracy [4], or extending those studies. Of note, when planning the numbers of PBMC to be frozen per cryovial, as a rule of thumb, one can anticipate recovery of ~50% of PBMC initially frozen after these cells are thawed and have undergone 5 days of polyclonal stimulation to promote terminal differentiation of resting B mem into ASC (see also Note 4).…”

“…Owing to the requirement to detect individual ASC-derived secretory footprints to accurately determine the frequency of antigen-speci c B cells, an ImmunoSpot® assay-related challenge to overcome is that B mem -derived ASC producing different classes and subclasses of Ig span orders of magnitude [4].…”

Assessing the affinity spectrum of the antigen-specific B cell repertoire via ImmunoSpot®

“…During the recent COVID pandemic we got reminded how rapidly following natural infection, or after vaccination, the induced speci c serum antibody levels can decline [15,16]. We also learned that frequently the detection of memory B cells (B mem ) is far more reliable for revealing whether an infection has occurred than measuring serum antibodies [17].…”

“…This permits "direct ex vivo" batch testing of samples irrespective of the time-point of their actual collection. By freezing the cells in aliquots, assays results can be reproduced with high accuracy [17],…”

“…14. In the initial test, it is suggested to plate PBMC (or other single cell suspensions) in serial dilution over a wide range of cell numbers per well because the frequencies of antigen-speci c ASC can span orders of magnitudes between individuals [17] and ASC producing different classes and subclasses of antigen-speci c (and total) Ig also span a wide range within an individual, and in the human population. In this way, not only the frequency of ASC can be reliably established by linear regression analysis [27] within the entire cell material tested, but the Goldilocks number for secretory footprints can be de ned as the maximal number of cells that can be plated while still being able to discern clearly individual secretory footprints.…”