Antibody levels poorly reflect on the frequency of memory B cells generated following SARS-CoV-2, seasonal influenza, or EBV infection

doi:10.21203/rs.3.rs-1792582/v1

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Antibody levels poorly reflect on the frequency of memory B cells generated following SARS-CoV-2, seasonal influenza, or EBV infection

https://doi.org/10.21203/rs.3.rs-1792582/v1

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Assessment of humoral immunity is commonly confined to measurements of serum antibody reactivity. These pre-formed immunoglobulin molecules can convey immunity by preventing a re-infection. In some instances, however, a re-infection does occur, and in such cases, it is the mobilization of an anamnestic B and T cell response that confers immune protection. Relying on fundamentally different mechanisms, therefore, 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 memory B cells reactive with the respective antigens. Weak correlations were found. The data suggest that 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 into 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.

Word Count = 166

Immunology

immune monitoring

antibody-mediated immunity

ELISPOT

ImmunoSpot

FluoroSpot

memory B cells

affinity maturation

SARS-CoV-2

Antibodies and memory B cells assume fundamentally different roles in mediating humoral immunity [1]. Antibodies constitute the “first wall” of host defense and can prevent re-infection with the same (homologous/homotypic) virus. Memory B cells constitute the “second wall”, and not only permit to mount an anamnestic antibody response against the homotypic virus (e.g., SARS-CoV-2 Wuhan-Hu-1 strain) after infection or vaccination, but also enable development of a faster and more efficient antibody response to emerging heterologous viral variants that can evade the “first wall” of pre-formed antibody (e.g. the Omicron variants of SARS-CoV-2). Therefore, assessment of memory B cells can provide insights into humoral immunity that measurements of serum antibodies do not convey.

The first encounter with a virus triggers a primary immune response with naïve antigen-reactive lymphocytes clonally expanding and their daughter cells differentiating into effector and memory cells. Effector cells contribute to instant host defense, while memory cells convey the potential to engage into an accelerated and more efficient secondary immune response in case the homologous virus or a heterologous variant strain is re-encountered later in life. For T and B lymphocytes alike, effector and memory cells have unique transcriptional profiles and life spans, and contribute fundamentally different functions towards maintaining host defense.

In the course of a primary B cell response, naïve B cells proliferate, undergo immunoglobulin class switching and affinity maturation while they differentiate into antibody-secreting cells (ASC, the effectors of the B cell system), or B memory (B_mem) cells. Plasma cells (PC) are the prevalent ASC in vivo. Plasma cells can be short- or long-lived and their survival depends on competition for niches in the bone marrow (BM) [2]. The antibody molecules they secrete have a relatively short half-life of around 3 weeks [3, 4]. Therefore, when antibodies are being monitored in serum (or in other bodily fluids), one needs to consider that the molecules detected are not long-lived remnants of immune memory but instead a reflection of ongoing secretory activity in the PC compartment. After some infections, exemplified by influenza, antibody production continues in spite of the elimination of the virus and high titers of virus-reactive antibodies can be detected for decades [5, 6]. After other infections, exemplified by SARS-CoV-2, virus-reactive antibody titers begin to decline within months [7–9]. The reason(s) why such differences in ASC activity occur are not well understood.

Antibodies are the secreted form of the B cell receptor (BCR) and convey a variety of effector functions [10, 11]. Most notably, antibody binding can directly neutralize an invading virus through binding to epitopes that prevent docking and/or entry into permissive host cells. Antibody binding, along with formation of antibody-antigen immune complexes, can also trigger activation of the complement cascade and release of pro-inflammatory cytokines, direct lysis of viral particles, and enhanced clearance of opsonized antigens by professional phagocytes. Furthermore, antibodies can selectively label virally-infected cells and target them for destruction through antibody-dependent cellular cytotoxicity (ADCC).

In addition to differentiation of ASC, long-lived B_mem cells are also generated during the course of a primary B cell response. Similar to their naïve precursor cells, B_mem cells are also resting lymphocytes that re-circulate via the blood throughout the lymphoid tissues of the body. Importantly, upon antigen re-encounter B_mem cells undergo rapid and robust proliferation and give rise to new generations of daughter cell progeny that include effector cells (ASC) and more B_mem cells. However, unlike their naïve precursor cells, B_mem cells are present in increased numbers in the body due to their clonally expanded status, and have already undergone immunoglobulin class switching and affinity maturation [12, 13]. Owing to this, the anamnestic response resulting from activation of pre-existing B_mem cells following re-encounter with a homologous virus is not only faster and more robust, but antibodies possessing increased affinity are also produced.

Even if variants of the virus are encountered against which the pre-existing antibodies are ineffective (as is the case with the Omicron variants of SARS-CoV-2 that acquired immune evasion mutations in the Spike protein circumventing the neutralizing capacity of pre-formed antibodies elicited through natural exposure and/or vaccination with the prototype (Wuhan-Hu-1) strain [14, 15]), the B_mem cells formed during the primary response are still likely to confer a critical immune advance owing to their semi-affinity matured and clonally expanded status. Specifically, even if these B_mem cells express a relatively low-affinity BCR towards the variant virus initially upon re-exposure, these cells can re-enter into germinal center (GC) reactions and acquire additional somatic mutations to refine and enhance their affinity towards the variant virus [16]: as the acquisition of somatic mutations is random, a subset of the B_mem cell repertoire elicited by the original SARS-CoV-2 infection, or through vaccination, is prone to possess an increased affinity for future antigenic variants. In this way, the immune response elicited following re-infection with an antigenically-related variant virus would not start from an unselected repertoire of naïve B cells, but from clonally expanded and partially affinity-selected B_mem cells. Where pre-formed antibodies fail to convey protection, B_mem cells could still stand a chance providing a second wall of defense [1].

The recent experience with SARS-CoV-2 vaccination further highlights the importance of distinguishing between immune protection conveyed by pre-formed antibodies versus protection conferred through recall of B_mem cells. Thus, while vaccination with Spike antigen induced a B and T cell response, it provided only short-term, or no reliable protection against becoming infected by SARS-CoV-2 variants: the pre-formed antibodies and effector cells could not completely prevent the virus from docking to and entering into the host’s cells to initiate viral replication. However, the severity of COVID is largely attenuated in vaccinated individuals conceivably due to their memory cells’ ability to rapidly re-engage into secondary heterosubtype-specific immune responses endowing a critical host defense advantage. Immune monitoring that was confined to measurements of serum antibody reactivity alone would therefore not suffice for predicting the vaccine’s efficacy without accounting for the memory cell compartment.

The presence of virus-reactive antibodies and their neutralizing potential can readily be measured using a number of well-established techniques [17–19]. In contrast, the detection and study of virus-reactive B_mem cells has been a major challenge, primarily because B cells reactive for any given antigen exist at very rare frequencies among all B cells, which themselves constitute only a fraction of all peripheral blood mononuclear cells (PBMC). Consequently, there is an abundance of publications on serum antibody reactivity and neutralizing capacity against SARS-CoV-2 [7, 9, 18, 20–23], but the number of successful B_mem studies are more limited for this virus [24–29], and other viruses as well [30–34]. Efforts to elucidate the antigen-reactive B_mem cell compartment have relied on the use of fluorescently-labeled antigens in conjunction with flow cytometry, assessment of antibody reactivity in culture supernatants from limiting dilution cultures of polyclonally stimulated PBMC, or on the ELISPOT/FluoroSpot technology. The strengths and weaknesses of these approaches for detecting antigen-reactive B_mem cells are outlined in Suppl. Table 1. Based on the compelling arguments in favor of ELISPOT/FluoroSpot, we selected to refine this technique so it becomes suitable for monitoring B_mem cells that are reactive with SARS-CoV-2, or other viral antigens.

In traditional B cell ELISPOT/FluoroSpot assays, the membrane is coated with an antigen of interest, e.g., a viral protein. Onto this antigen-coated membrane, the test subject’s PBMC are seeded containing the rare antigen-reactive ASC of interest. In the absence of recent antigen encounter, however, B_mem cells exist in a resting state and do not constitutively secrete antibody. To overcome this obstacle, PBMC need first to be activated through polyclonal stimulation to drive terminal differentiation of B_mem cells into ASC prior to their measurement in the assay [35–37]. While most B cells can be transitioned into ASC following polyclonal stimulation protocols, each secreting large quantities of its encoded BCR as soluble antibody, only those ASC producing antibodies with sufficient binding affinity for the coated antigen will leave a detectable antibody foot-print on the antigen-coated assay membrane. As such, the number of spot-forming units (SFU) in a well reveals the number of antigen-reactive ASC present in the well versus all ASC present in that well. In this way, the magnitude of the antigen-reactive B_mem cell compartment can be established. The immunoglobulin class and subclass produced by each ASC can also be defined through usage of class/subclass-specific detection reagents [38]. The visualization of the plate-bound analytes relies either on the use of precipitating substrates (ELISPOT) or on fluorescence (FluoroSpot). As other than the detection step ELISPOT and FluoroSpot are essentially the same, jointly we refer to them as ImmunoSpot®.

The classical B cell ELISPOT assay was introduced decades ago [39, 40], but since then has only been implemented successfully for few human immune monitoring efforts [41] due to the simple reason that for most antigens such assays could not be established. This is because absorption of the antigen to the membrane is governed by weak, non-specific binding forces such as hydrophobicity and charge. Successful coating therefore depends on the physical properties of the membrane chosen versus the corresponding properties of the antigen itself. To overcome this limitation in development of B cell ImmunoSpot® assays for “any antigen of interest”, we recently introduced an approach termed “affinity capture coating” (ACC) in which improved antigen absorption to the membrane is achieved through pre-conditioning the assay membrane with an antibody specific for the genetically-encoded hexahistidine (6XHis) affinity tag of recombinant proteins [42] enabling the subsequent high-affinity capture of any 6XHis-tagged protein. Using this universal ACC approach, we developed ImmunoSpot® assays that enabled the detection of memory B cells reactive with antigens representing SARS-CoV-2, seasonal influenza, and Epstein-Barr virus (EBV) [42]. Suppl. Figure 1 illustrates the ACC ImmunoSpot® approach utilized in this study for detecting rare antigen-reactive B_mem cells against recombinantly expressed viral antigens.

Access to the above antigen-specific B cell ImmunoSpot® assays permitted us in the present study to ask a fundamental question that (due to the limitations in detecting antigen-reactive B_mem cells so far) has not been systematically addressed: how do antibody levels reflect on B_mem cell frequencies reactive with a given antigen? In other words, do high antibody levels in an individual indicate that this person also has high numbers of B_mem cells as intuition would suggest? Is therefore the assumption warranted that, if an individual is more protected from becoming re-infected owing to his/her higher titer of pre-existing antibodies than another individual who developed a lower antibody titer, is the former also prone to mount a stronger anamnestic antibody response following re-infection? Do low/negative antibody titers in an exposed/vaccinated individual imply that this individual did not possibly develop an even stronger B cell memory response than an individual with high antibody titers?

We studied here the relationship between antibody levels and B_mem cell frequencies for viral infections that fundamentally differ in terms of verifiable exposure. One cohort consisted of unvaccinated subjects that were infected with the SARS-CoV-2 virus, and these samples were collected after complete convalescence. A second cohort consisted of subjects whose plasma and PBMC had been cryopreserved in the pre-COVID era (prior to November 1, 2019); the latter unvaccinated subjects serve as an unambiguous negative control group for SARS-CoV-2 immunity. The importance of the latter control group is highlighted by the fact that for the other viruses we studied (influenza or EBV) unambiguous negative controls cannot be established as most adults in the human population have been exposed to them, and the “positive/negative” judgment call has been based solely on serum antibody levels present in these subjects. Calling into question whether antibody reactivity is a reliable indicator of antigen exposure and immunity, in a previous study [43], we established that the majority of human cytomegalovirus- (HCMV) seronegative donors indeed have high numbers of HCMV antigen-reactive CD4⁺ and CD8⁺ memory T cells, as well as B_mem cells. The present study will confirm and extend this notion.

The three virus infections we studied here for the relationship between antibody levels and B_mem cell frequencies also fundamentally differ in terms of the duration and periodicity of antigen exposure. SARS-CoV-2 infection results in clearance of virus by the time of recovery from the infection, typically within two weeks. Therefore, the subjects in our SARS-CoV-2 cohort had a single, full-blown exposure to this virus without any subsequent antigen stimulation. EBV, in contrast, is not cleared following resolution of the acute infection and instead persists latently in B cells causing occasional subclinical viral flares and repetitive, and potentially chronic, antigen stimulation [44, 45]. Influenza, like SARS-CoV-2, is also rapidly cleared following initiation of a successful immune response. However, the influenza exposure histories of our donor cohorts, and specifically their circulating antibody and antigen-reactive B and T cell repertoires, are far more complex owing to the contributions of prior infections and/or vaccinations over their respective lifetimes [46, 47].

Studying for these three fundamentally different viruses and immune scenarios the respective antigen-reactive antibody levels by ELISA, and the B_mem frequency in PBMC by ImmunoSpot®, we show that in each of these antigen systems these two immune parameters do not strongly correlate. Discordance between pre-formed humoral immunity and B cell memory potential, therefore, might be common in anti-viral immunity.

2.1. Human Subjects

PBMC and plasma samples from pre-COVID-19 era donors were obtained from healthy adults and originate from CTL’s ePBMC® library (CTL, Shaker Heights, OH, USA). Samples (n = 54) were collected prior to November 1, 2019 at FDA-registered collection centers from IRB-consented healthy human donors by leukapheresis and then were sold to CTL identifying donors by code only while concealing the subjects´ identities. PBMC were cryopreserved and stored in liquid nitrogen until testing. Plasma was aliquoted and stored at -20˚C until testing. Additional information pertaining to the pre-COVID-19 donors is provided in Suppl. Table 2.

PBMC and plasma samples comprising the convalescent COVID-19 donor cohort (n = 25) were obtained from three sources as follows: five donors were purchased from Oklahoma Blood Institute (Oklahoma city, OK) and were received as previously cryopreserved PBMC and plasma aliquots; ten donors were recruited by American Red Cross (Atlanta, GA, USA), BioIVT (Westbury, NY), or Stem Express (Folsom, CA) with IRB approval and then were sold to CTL identifying donors by code only while concealing the subjects’ identities. Ten donors were collected internally at CTL under an Advarra Approved IRB #Pro00043178 (CTL study number: GL20-16 entitled COVID-19 Immune Response Evaluation). All PBMC were stored in liquid nitrogen until testing. Plasma was aliquoted and stored at -20˚C until testing. All subjects included in the COVID-19 cohort tested positive for SARS-CoV-2 RNA by PCR. Additional information on the COVID-19 donors is provided in Suppl. Table 3.

2.2. Polyclonal B Cell Stimulation

Detailed methods of thawing, washing and counting of PBMC have been previously described [48]. The freshly thawed PBMC samples were resuspended in B cell medium (BCM) containing RPMI 1640 (Lonza, Walkersville, MD) supplemented with 10% fetal bovine serum (Gemini Bioproducts, West Sacramento, CA), 100 U/mL penicillin, 100 U/mL streptomycin, 2 mM L-Glutamine, 1mM sodium pyruvate, 8mM HEPES (all from Life Technologies, Grand Island, NY) and 50 µM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). PBMC were then stimulated with Human B-Poly-S (CTL) at 2 x 10⁶ cells/mL in 25 cm² tissue culture flasks (Corning, Sigma-Aldrich) and incubated at 37°C, 5% CO₂ for five days to drive differentiation of resting memory B cells into ASC prior to evaluation in ImmunoSpot→ assays.

2.3 Recombinant Proteins

SARS-CoV-2 Spike subunit 1 (S1) was purchased from Creative Diagnostics (Shirley, NY, USA). SARS-CoV-2 Nucleocapsid (NCAP) was purchased from RayBiotech (Peachtree Corners, GA). Recombinant Epstein-Barr virus nuclear antigen 1 (EBNA1) protein was purchased from Serion (Würzburg, Germany). Recombinant hemagglutinin (rHA) proteins encoding A/California/04/2009 (CA/09, H1N1), A/Texas/50/2012 (TX/12, H3N2) and B/Phuket/3073/2013 (Phuket/13, FluB) seasonal influenza vaccine strains were acquired from the Center for Vaccines and Immunology (CVI) (University of Georgia (UGA), Athens, GA, USA) and have been described previously [49, 50]. Importantly, all recombinant proteins used in this study possessed a genetically-encoded 6XHis affinity tag.

2.4. B Cell ImmunoSpot® Assays

Following polyclonal stimulation, PBMC were harvested from tissue culture flasks and washed with PBS prior to counting using CTL’s Live/Dead cell-counting suite on an ImmunoSpot® Ultimate S6 Analyzer. Cell pellets were resuspended at 1–3 x 10⁶ live cells per mL (when measuring antigen-reactive IgG⁺ ASC) or 3 x 10⁵ live cells per mL (for detection of all IgG-secreting cells) in complete BCM and seeded into ImmunoSpot® assays.

For enumeration of all IgG-secreting cells (total IgG⁺ ASC), the cell suspensions were serially diluted 2-fold in duplicates, starting at 3 x 10⁴ live cells/well, in round-bottom 96-well tissue culture plates (Corning, Sigma-Aldrich). Subsequently, cells were transferred into ImmunoSpot® assay plates that were pre-coated with anti-κ/λ capture antibody reagents (from CTL) and incubated for 16 h at 37°C, 5% CO₂. Plate-bound immunoglobulin (Ig) spot-forming units (SFU) were subsequently visualized using a human IgG-detecting ImmunoSpot® kit (from CTL) according to the manufacturer’s instructions.

For enumeration of antigen-reactive IgG⁺ ASC, ImmunoSpot® assays were performed with ACC as previously described [42]. Briefly, assay plates were first pre-conditioned with 70% (v/v) EtOH followed by two washing steps with PBS. Next, wells were coated with purified anti-6XHis tag antibody (BioLegend) at 10 µg/mL in PBS overnight at 4°C. The following day, assay plates were washed once with PBS and then coated overnight with 6XHis-tag labeled recombinant protein at 10 µg/mL in PBS. After overnight incubation of the 6XHis-tagged recombinant protein coating solutions at 4°C, plates were washed once with PBS and then blocked with complete BCM for 1 h at room temperature prior to addition of polyclonally-stimulated PBMC at the specified cell numbers per well. Plates were incubated at 37°C, 5% CO₂ for 16 h and SFU were subsequently visualized using the human IgG-detecting ImmunoSpot® kit (from CTL) according to the manufacturer´s instructions.

2.5. ImmunoSpot® Image Acquisition and SFU Counting

Plates were air-dried prior to scanning on an ImmunoSpot® Ultimate S6 Analyzer. Total or antigen-reactive IgG⁺ SFU were then enumerated using the Fluoro-X suite and Basic Count mode of ImmunoSpot® Software (Version 7.0.27). To account for the variable starting frequencies and differentiation of memory B cells during the polyclonal stimulation protocol, data are reported as the frequency of antigen-reactive ASC among total IgG⁺ ASC [51]. As ImmunoSpot® B cell kits, analyzers and software proprietary to CTL were used in this study; we refer to this collective methodology as ImmunoSpot®.

For accurate counting of SFU per well we relied on a serial dilution strategy shown in Fig. 1A and Suppl. Figures 2–4, and detailed in Results and Discussion. As shown, at low SFU numbers per well there was a linear relationship between cell numbers plated and SFU counted, from which, via linear regression and extrapolation, the frequencies of ASC in PBMC could be established. For routine testing our ImmunoSpot® assay approach was tailored to achieve maximal sensitivity in detecting low frequency memory B cell responses, and thus a single (1–3 x 10⁵) cell per well input was used. In such cases, SFU counts in the critical < 100 SFU/well range are accurate; however, higher SFU counts per well approach the “upper bound” of accurate enumeration and therefore likely underrepresent the actual antigen-reactive B cell frequency.

2.6 Bivariate Visualization of FluoroSpots

Counted FluoroSpots from replicate wells of an individual donor originating from the same cell input were merged into flow cytometry standard (FCS) files using the ImmunoSpot® Software (Version 7.0.27.0) and visualized using Flowjo^™ (Version 10.6.2) (Ashland, Oregon USA).

2.7. ELISA Assays

To evaluate IgG reactivity in plasma samples, Immulon→ 4HBX plates (Thermo Fisher Scientific, Waltham, MA) were coated overnight at 4°C with 2 µg/mL of the respective recombinant antigens: rHA representing CA/09, TX/12 or Phuket/13 of seasonal influenza strains or EBNA1 protein in carbonate buffer (pH 9.4); SARS-CoV-2 S1 or SARS-CoV-2 NCAP proteins in PBS. After blocking the plates with ELISA blocking buffer containing 2% v/v BSA in PBS with 0.1% v/v Tween20 (Sigma-Aldrich) for 1 h at room temperature, plasma samples were serially diluted and incubated for 2 h. Following four washes with 150 µL of PBS, horseradish peroxidase-conjugated anti-human IgG detection reagent (from CTL) was added to the plates and incubated for 1 h at room temperature. After washing the plates four times with PBS, 100 µL of TMB chromogen solution (Thermo Fisher Scientific, Waltham, MA) was added to develop the assay. Conversion of the TMB substrate was terminated by addition of 100 µL/well 1M HCl and optical density was measured at 450nm (OD₄₅₀) using a Spectra Max 190 plate reader (Molecular Devices, San Jose, CA). The abundance of IgG reactivity against the respective antigens was then interpolated into µg/mL IgG equivalents using SpotStat™ (Version 1.6.4.0, CTL) based on standard curves generated by directly coating decreasing quantities of a reference IgG preparation (from Athens Research and Technology, Athens, GA) in duplicate into designated wells of each assay plate.

2.6. Statistical Methods

Student’s t-tests were used to evaluate differences in serum antibody reactivity or antigen-reactive B cell frequencies between pre-COVID-19 and COVID-19 donor cohorts (GraphPad Prism Version 9.2, San Diego, CA). Pearson correlation analysis was performed using GraphPad Prism software on log-transformed antigen-reactive ASC data and the corresponding antigen-reactive IgG titers (expressed as µg/mL IgG equivalents). Regressions were also performed using GraphPad Prism and the R² values, 95% confidence bands and p-values less than 0.05 are plotted in the corresponding figures.

The primary goal for this study was to test the hypothesis that virus-reactive antibody levels are reflecting on the frequency of B_mem cells. Based on long held “textbook knowledge”, such a close correlation could be expected, as (a) plasma cells are thought to be long-lived and continuously produce antibody, (b) B_mem cells are also assumed to be long-lived, and (c) both cell types were thought to arise during the course of a B cell immune response at a constant ratio from a common precursor cell. The recent literature, discussed below, including the data presented in the following communication, call into question that such a direct correlation would be the rule after immune responses to viral infections.

Our first concern addressing this hypothesis was to optimize the accuracy of measuring both antigen-reactive antibody levels and the frequency of antigen-reactive B_mem cells. For the former, we performed standard ELISAs involving serial dilutions of the donor plasma samples in conjunction with a “µg/mL equivalent scale” for analyzing the data [52–55]. This approach is superior to the “area under the curve” approach as it leverages an internal (plate-specific) reference standard and thus is independent of assay-associated variability related to development time and temperature.

While the methodology for the quantification of antigen-reactive antibodies in plasma by ELISA is well-established, we first needed to develop such for the objective and accurate counting of antigen-reactive B_mem cells in the blood. Empirically, antigen-reactive B cell ImmunoSpot® assays result in diverse spot morphologies [42] (see also Figs. 1B and 1C). This outcome is expected, as the secretory foot-prints of individual ASC is defined by a multitude of parameters [56], including (a) the net amount of antibody produced by the individual B cells during the assay’s duration, (b) the kinetics of antibody production by the ASC, and (c) the functional affinity of each ASC’s antibody for the antigen (that can encompass a broad spectrum among an antigen-reactive B cell repertoire defining the capture and dissociation rates of antibody binding to antigen). Furthermore, pan-well or regional “ELISA” effects modulate the background membrane staining when antibody that “escaped” into the supernatant is captured distally from the source ASC. Crowding of spots also interferes with unambiguous counting. For all these reasons, the first part of this manuscript is dedicated to establishing unambiguous frequencies of antigen-reactive B cells in PBMC.

3.1. Establishing unambiguous frequencies of antigen-reactive B cells in PBMC

The accurate detection and counting of antigen-reactive B cells in PBMC predicts a linear relationship between the cell numbers plated into the ImmunoSpot® assay, and the antigen-reactive SFU counted. This is because B cells are the only cell type that secrete antibodies, and because each B cell produces antibody with pre-defined specificity. For each antigen reported in this study, we have established the ideal test conditions for accurate frequency measurements. PBMC have been polyclonally activated with R848 and IL-2 for five days to promote the differentiation of resting B_mem cells (which do not secrete antibody) into ASC that can be detected in ImmunoSpot® assays via their secretory foot-prints [35–37]. Such pre-stimulated PBMC were plated in serial dilution into antigen-coated assay plates while increasing the number of replicate wells at lower cell numbers to account for the expected Poisson variation. Suppl. Figure 2 shows raw data for such experiments; in the example shown two representative donors that have recovered from COVID-19 were evaluated for ASC reactivity against the SARS-CoV-2 S1 protein.

As expected, the number of detectable SFU decreased with the number of stimulated PBMC plated (Fig. 1A and Suppl. Figure 2A). However, secretory foot-prints of individual ASC were not readily resolved at the highest cell inputs due to the confluence of spots and elevated background membrane staining arising as a consequence of the elevated antigen-reactive ASC frequency. As expected, at lower cell inputs the foot-prints of individual ASC became clearly discernable and facilitated accurate enumeration of individual antigen-reactive ASC in such wells (Fig. 1B). Also as expected, the SFUs displayed a wide spectrum of sizes and fluorescent intensities (Figs. 1B and 1C) reflecting on the individual ASC’s range of productivity and functional affinity for the SARS-CoV-2 S1 protein. Therefore, when we used machine reading for the enumeration of SFU, we counted all SFU, including all sizes and densities. Counting of such “ungated events” showed a close to perfect linear relationship between SFU counts and cell numbers plated per well for (but only for) wells that contained low numbers of SFU: for COVID-19 Donor 1, for example, these were wells containing ≤ 2 x 10⁴ PBMC/well (Fig. 1E). For this donor, SFU counts for cell numbers exceeding 2 x 10⁴ PBMC/well were reduced below the expected count (Fig. 1D). Once the frequency-dependent linear range has been established for a subject, linear regression permitted to reliably calculate the frequency of antigen-reactive B cells within the PBMC: for COVID-19 Donor 1 this number was computed to be 195 SARS-CoV-2 S1-reactive ASC/10⁵ PBMC, for COVID-19 Donor 2, providing another example, it was 163 S1-reactive ASC/10⁵ PBMC (Suppl. Figure 2B).

The above data establish that accurate counting of antigen-reactive B cells in ImmunoSpot® assays requires to establish the range in which SFU counts and cell input per well exhibit a linear relationship from which frequencies can be reliably extrapolated. This is critical as the numbers of antigen-reactive B cells can span several orders of magnitudes even between antigen-primed individuals (see below). We optimized test conditions, and verified these basic assumptions for the accurate counting of SFUs for all antigen-reactive B cell ImmunoSpot® assays reported in this publication: representative results from such experiments are shown in Suppl. Figures 2–4.

As a positive control, we also performed total IgG⁺ ImmunoSpot® assays in parallel to verify the functionality of B cells following their in vitro stimulation. In this assay variant, the membrane is coated with anti-human immunoglobulin light chain (IgL) (anti-Igκ/Igλ) capture antibody instead of the antigen itself, capturing the secretory foot-print of all ASC irrespective of their binding reactivity. Applying the rules described above we have established the frequency of total IgG⁺ ASC in each test subject through serial dilution (the raw data and results for six representative samples are depicted in Suppl. Figure 5). Because the frequency of total IgG⁺ ASC exhibited considerable inter-individual variation among the test subjects following in vitro stimulation, when frequencies of antigen-reactive IgG⁺ ASC are reported in the following, they are expressed as a percentage of the total IgG⁺ ASC compartment, i.e., “% antigen-reactive IgG⁺ ASC” [51].

3.2. Equal overall assay performance of pre- and post-COVID-19 PBMC

SARS-CoV-2 exposure, unlike the endemic seasonal influenza and EBV infections also studied here, is unique in as much that the exposure can be verified beyond measuring antibody reactivity. As the first laboratory confirmed case of SARS-CoV-2 virus infection in the United States occurred on January 20, 2020 [57], PBMC collected before November 1, 2019 have been verifiably derived from subjects who could not have been exposed to SARS-CoV-2, and thus must be immunologically naïve to this virus. It is a matter of debate, however, how much T and B cell cross-reactivity exists in such pre-COVID-19 era subjects elicited through prior exposure(s) to endemic coronavirus strains [58–61], and possibly other antigens [62]. It is also remains unclear whether such pre-existing, cross-reactive immunity contributes to defining the severity of the primary SARS-CoV-2 infection. Also, unlike for influenza and EBV, not only exposure itself but even the time-point of the primary SARS-CoV-2 infection can be verified by PCR testing. For this study we therefore could compare B cell reactivity to SARS-CoV-2 antigens in two cohorts that are highly defined with respect to immune exposure to this virus: PBMC collected from individuals who could not have been infected (the “pre-COVID-19” cohort), and those who have been collected after PCR-verified SARS-CoV-2 infection during the first wave, in 2020, caused most likely by the original “prototype” Wuhan-Hu-1 strain (the “COVID-19” cohort). Notably, all PBMC were collected before vaccination became available.

PBMC of the test subjects were cryopreserved according to a protocol that maintains B cell functionality [63]. As shown in Suppl. Figure 6A, the viability of the PBMC was comparable for both cohorts after thawing. So was the functionality: the number of total IgG⁺ ASC, while showing the expected inter-individual variation within each group was comparable among the two cohorts (Suppl. Figure 6B). When tested in two separate experiments, the frequencies of total IgG⁺ ASC reproduced well for the individual donors (Suppl. Figure 6C) suggesting that the inter-donor variability seen is inherent to each donor, and does not represent an assay variable. Suppl. Figure 6D depicts the correlation between PBMC viability after five days of polyclonal stimulation for each donor in both cohorts plotted versus the total IgG⁺ ASC frequency (that is, “what percentage of viable cells were IgG⁺ ASC”). While, as expected, there was no direct correlation between overall viability of the PBMC and total IgG⁺ ASC frequency, the cells from both cohorts behaved alike. Collectively, these observations suggest that if any difference is seen in antigen-reactive frequencies among these cohorts that it cannot be attributed to the freezing conditions or the duration of time the cells were stored under liquid nitrogen. (The comparison of influenza- and EBV-reactive B cell frequencies between the two cohorts below, in Fig. 4, and the inter-assay reproducibility of both total and antigen-reactive IgG⁺ ASC frequencies, presented in Suppl. Figures 6C and 7, respectively, will further substantiate this claim).

3.3. Exquisite specificity of SARS-CoV-2 antigen-reactive B cell ImmunoSpot® assays

With the exception of a single donor, PBMC from all other subjects in the COVID-19 cohort (n = 25), collected following convalescence from SARS-CoV-2 infection, displayed S1-reactive IgG⁺ ASC after polyclonal stimulation, albeit in frequencies that spanned three orders of magnitude, ranging between 0.01–1.15% of all IgG⁺ ASC (Fig. 2A). In contrast, less than 5 S1-reactive IgG⁺ ASC were detected in any of the pre-COVID-19 era donors, even at the highest cell number tested (3 x 10⁵ PBMC/well). Importantly, at this high cell input such S1-reactive IgG⁺ ASC were often too numerous to accurately count for several COVID-19 donors (Suppl. Figure 2).

Testing for B_mem cell reactivity against the SARS-CoV-2 nucleocapsid (NCAP) protein reproduced the exquisite specificity seen for the S1 antigen (Fig. 3A). As with the S1 antigen, NCAP-reactive ASC occurred in frequencies less than 5 SFU per 3 x 10⁵ PBMC for all pre-COVID-19 era donors tested. In stark contrast, IgG⁺ ASC with reactivity against seasonal influenza and/or EBV antigens were readily detectable in pre-COVID-19 donors (Fig. 4 and Suppl. Table 4). This absence of detectable NCAP-reactive IgG⁺ ASC in pre-COVID-19 donors is of particular interest, firstly, because the NCAP protein expressed by novel SARS-CoV-2 virus strains share more sequence conservation and predicted immunogenic epitopes with circulating seasonal coronaviruses than the Spike (S1) protein and as such is more likely to cross-react with B cells triggered by seasonal coronaviruses [64, 65], and second, because establishing immunity to NCAP is gaining importance for the immunodiagnostic of SARS-CoV-2 infection in COVID-19 vaccinated (i.e., Spike antigen immunized) individuals.

3.4. Discordance between SARS-CoV-2 antigen-reactive memory B cell frequencies and antibody levels

We also performed ELISA assays involving the same recombinant SARS-CoV-2 antigen preparations as applied above for ImmunoSpot® testing. Plasma from both cohorts were tested in serial dilutions alongside an IgG reference standard to establish for each subject the concentrations of S1 (Fig. 2B) and NCAP (Fig. 3B) antigen-reactive IgG levels. Judged as cohorts, the results were clear-cut and revealed significantly increased IgG reactivity against the SARS-CoV-2 S1 and NCAP coating antigens. However, the results were less clear-cut when judged at the level of individual subjects. Approximately half of the subjects in the COVID-19 cohort showed low levels of IgG reactivity against either the S1 or NCAP coating antigens while displaying elevated frequencies of antigen-reactive IgG⁺ ASC (derived from memory B cells) following in vitro stimulation (Figs. 2C and 3C). Thus, for immunodiagnostic purposes, detection of B cell memory appears to be a more sensitive and reliable indicator of SARS-CoV-2 infection history than measurements of antibody reactivity alone.

For both the SARS-CoV-2 S1 and NCAP antigens, plasma IgG levels and frequencies of antigen-reactive IgG⁺ ASC (derived from B_mem cells recirculating in blood) were poorly correlated. Specifically, the R² values were 0.2535 for the S1 protein (Fig. 2C) and 0.0155 for NCAP (Fig. 3C), respectively. Therefore, in the context of SARS-CoV-2 immunity, plasma antibody levels were poor indicators of the corresponding antigen-reactive memory B cell pool sizes. These measurements were made on blood samples collected within months after recovery from COVID-19. As antibody titers are lower after mild- compared to severe SARS-CoV-2 infection [66, 67], and additionally wane over time [7–9], while B_mem cell frequencies are thought to be more stable over time, one might expect the discordance to grow as time passes, but longer-term longitudinal studies will be needed to address this point. Irrespective of the outcome of those future studies, however, the main message of this communication is likely to hold up: detection of B_mem cells themselves is likely to be a more sensitive and reliable indicator of immune exposure and memory potential than standard measurements of antibody reactivity alone, and antibody levels are poor indicators of memory B cells frequencies in any given individual.

3.5. Discordance between influenza virus and EBV antigen-reactive memory B cell frequencies and antibody levels

In the next set of data, we aimed to establish whether the findings reported above for SARS-CoV-2 are unique to this viral infection and/or the circumstances of our testing. The IgG⁺ B cell response induced by SARS-CoV-2 infection might be unique due to the immune evasion strategies of this virus [68]. But even if that is not the case, the dissociation between plasma antibody levels and B_mem cell frequencies could just be a feature of (a) a primary B cell response seen (b) shortly after recovery from a mild infection with a virus that (c) typically is cleared within 2 weeks, as these all apply to the COVID-19 cohort tested above. Studying influenza-reactive B cell immunity might therefore provide further insights in this regard. The influenza virus is also cleared by the immune system (with the rare exceptions of severe infections) within 2 weeks post-infection. However, the time-point of the primary infection likely lies in the distant past, years or even decades ago, and re-infections causing secondary B cell responses can be assumed for most adults. As seen in Figs. 4A-C, the correlation between plasma antibody levels and B_mem cell frequencies were only marginal for three representative influenza virus strains, with R² values of 0.1896 for CA/09 (H1N1), 0.0789 for TX/12 (H3N2), and 0.0913 for Phuket/13 (FluB) rHA antigens, respectively. These data suggest that the observations reported above for SARS-CoV-2 are neither unique to this virus, nor related to the early time-point of measurements, but might be a more universal feature of the B cell response following (at a minimum) respiratory tract infections.

Infection with EBV represents yet a fundamentally different immunological scenario. The primary infection occurs mostly during young adulthood (EBV infection is also called “student kiss fever”), and is systemic. Furthermore, unlike SARS-CoV-2 and influenza, EBV is not cleared from the body but persists lifelong with occasional re-activation episodes, and EBNA1 is expressed even in latency [69]. Thus, the B cell response to EBNA1 can be considered a prototype for that occurring in the immune scenario of systemic antigen persistence. As seen in Fig. 4D, the correlation between EBNA1-reactive IgG antibody levels in plasma and IgG⁺ memory B cell frequencies was only marginal in healthy adult donors, with an R² value of 0.1732. Also important for immune diagnostic purposes, like with SARS-CoV-2 and influenza, several subjects exhibited low levels of plasma antibody reactivity against the EBNA1 protein, a finding that when viewed in isolation could be suggestive of either a lack of virus exposure or for developing a weak B cell response to the exposure, yet many of these donors possessed B_mem cells in high frequency. Collectively, the findings made for EBNA1 further support the notion that monitoring the B cell memory compartment itself can be a more sensitive and reliable indicator of immune exposure and memory potential than standard measurements of antibody reactivity alone.

3.6 High reproducibility of total and antigen-reactive IgG⁺ ASC frequency measurements

While we had limited numbers of PBMC available for most subjects in the COVID-19 cohort, cells obtained from pre-COVID-19 era donors were available in sufficient quantities to assess the reproducibility of either total or antigen-reactive IgG⁺ ASC frequency measurements. We tested replicate aliquots of PBMC cryopreserved from the same blood draw in two separate experiments. Thus, except for the cryopreserved cell material being the same, all other steps of the testing process were independent assay variables, including (a) thawing and washing of the cryopreserved cells, (b) live/dead cell counting and adjusting the PBMC concentration to 2 x 10⁶ cells/mL for the (c) subsequent polyclonal stimulation during the five day cell culture period, followed by renewed live/dead counting of the PBMC and re-adjusting their concentration for being plated into (d) the FluoroSpot assays (total or antigen-specific). The (e) visualization and (f) counting of the SFU also represent potential inter-assay variables. In this way, for each pre-COVID-19 era donor, frequencies for (g) total IgG⁺ ASC, as well as (h) the frequency of the antigen-reactive IgG⁺ ASC were determined, permitting to calculate “% antigen-reactive IgG⁺ ASC relative to total IgG⁺ ASC”. Suppl. Figure 6C summarizes the results obtained from repeat testing of total IgG⁺ ASC frequencies using replicate vials of PBMC while Suppl. Figures 7A-C depict the reproducibility in antigen-reactive IgG⁺ ASC frequencies established against the CA/09 rHA, TX/12 rHA and EBNA1 proteins. Collectively, these data showed that, in spite of the multitude of potential assay variables, the frequencies of ASCs reactive with these antigens reproduced closely.

3.7. Discussion of the mechanism underlying discordance and implications

Our current understanding of the fate decision checkpoints determining whether an activated B cell will join the B_mem cell compartment or differentiate into an antibody-secreting PC cell is the subject of several excellent reviews [1, 16, 70–73]. While many parameters can contribute to B cell fate decisions after antigen encounter, in the following we will focus on just two mechanisms that could account for, or contribute to the discordance between circulating antibody levels and antigen-reactive B_mem cell frequencies.

First, limitations on PC versus B_mem cell survival might account for our finding. PC can be classified into two types; those that are short-lived and contribute to antibody titers only acutely, and those that are long-lived PC (LLPC) and provide sustained antibody production for decades and potentially the lifetime of an individual [74–76]. Presently there is an incomplete understanding of what distinguishes the ability of a LLPC to survive relative to a short-lived ASC [2]. Importantly, LLPCs are not intrinsically long-lived and instead their survival is dependent on the acquisition of a distinct transcriptional profile and their ability to access specialized and pro-survival niches such as those existing in the BM. Furthermore, there is likely only a finite number of suitable niches in such anatomical locations in which PCs can take up residence and acquire longevity through their intimate interactions with stromal cells and receipt of pro-survival cues [2, 77, 78]. As such, a plausible explanation for the reduced levels of antigen-reactive circulating antibody detected in many of the subjects investigated in this communication is that GC-derived ASCs were in fact generated as a consequence of virus infection, but failed to successfully take up long-term residence in the BM. B_mem cells, in contrast, do not need to compete for such niches for their survival. While in theory the ImmunoSpot® assay would be particularly well-suited to directly test this hypothesis by enumerating in individual subjects’ PC frequencies in BM [79] versus B_mem cell numbers in the blood, the BM compartment is not readily accessible for routine immune monitoring and thus assessment of LLPC in our donor cohort was not possible. While this mechanism might explain why antibody levels can be low in the presence of abundant B_mem cells, it does not account for the reverse scenario.

The differential, affinity-based selection of mutated B cell subclones along the PC cell versus the B_mem cell differentiation pathways might account for our findings. The GC is an anatomical site in which antigen-activated B cells can undergo multiple rounds of cell division and progressively acquire somatic hypermutations in their BCR’s IgH/IgL. Perhaps most relevant to the interpretation of the data presented here is the well-established notion that the fate decision of a given GC B cell subclone is determined based on the affinity of its BCR for the eliciting antigen [1, 16]. Namely, GC B cell subclones endowed with a low-affinity BCR for the antigen stop proliferating (especially at later stages of the response when antigen becomes limiting) due to insufficient interactions with follicular T helper (T_FH) cells, and instead exit the GC reaction and become long-lived B_mem cells. In contrast, GC subclones possessing a high-affinity BCR are instructed by T_FH cells to undergo further rounds of proliferation and acquire additional somatic hypermutations [80, 81]. As antigen become increasingly more limiting in the GC reaction, the pressure for selection of high-affinity GC B cell subclones becomes even more stringent while the remainder of B cells with lower affinity for the antigen are shunted towards the B_mem cell pool. This affinity-based selection process, often referred to as affinity maturation, ultimately leads to the differentiation of PC producing antibody with an exquisitely high-affinity for the eliciting antigen and B_mem cells of high, but primarily lower affinity [82]. Consequently, GC-derived PC and B_mem cells represent two related, yet non-overlapping, antigen-reactive repertoires with divergent cell fate decisions based on their BCR’s affinity for antigen.

Therefore, it is assumed that B cell responses that give rise to GC reactions build “two walls of protection against pathogens” [1]. The first wall, encompassed by pre-formed antibodies possessing high affinity afford the advantage of providing instant defense against the homologous pathogen, but on the downside, these antibodies can potentially interfere with the ability to respond to new variants that are antigenically similar [83, 84]. The second wall of defense is comprised by B_mem cells that not only can participate in anamnestic responses against the homologous virus, but also contribute clonally expanded, class-switched, and semi-affinity matured precursor cells that may be reactive with emerging variants of the pathogen and which can undergo further affinity maturation to fine tailor the B cell response to such variants [85, 86].

The above notion of divergent affinity-based selection underlying alternative PC cell versus B_mem cell repertoires has primarily arisen from studies of murine models involving, by necessity, minimalist approaches like the use of BCR-transgenic mice and model antigens [87]. To what extent they are applicable to human B cell responses against viruses in large remains unverified. These murine studies were further enabled by reliance on variable-reducing test conditions, including the homogenous genetic background, age, and sex of the mice studied, their controlled specific-pathogen free environment, defined antigen exposure, and through unfettered access to the lymphoid tissues in which B cell immune responses evolve. As indicated previously, human PC residing in the BM are not readily accessible for general immune monitoring purposes. While human B_mem cells can be readily accessed via blood draws, the ability to study them systematically in larger cohorts awaited an enabling technology. To our knowledge the data communicated here represent the first systematic study comparing the frequency of virus-reactive B_mem cell frequencies with circulating antibody levels in sizable human cohorts. The basic findings we report here, that antibody levels and B_mem cell frequencies are frequently discordant, may not come as a surprise in face of the murine literature, however, the extent of inter-individual variation between serum antibody and B_mem cell frequencies we observed in our human cohorts was, to a degree, unexpected. All of our PBMC donors were healthy young or middle-aged adults and all the B cell responses we studied were induced by, and directed against, viruses that these adults successfully controlled. And yet, in some of these individuals, memory B cell frequencies were remarkably high while antibody titers were surprisingly low, and vice versa. We postulate here that there might be an evolutionary pressure behind developing such discordant phenotypes.

With fundamental pros and cons for pre-existing, antibody-afforded versus B_mem cell-mediated protection, there could exist an evolutionary advantage for endowing different individuals in the population with the propensity to preferentially rely on one or the other immune defense strategy. While with the viruses we studied here either strategy is compatible with successful defense, it can be envisioned that with some pathogens the fate decisions of B cells could have profound implications on the outcome of the host.

3.8. Concluding remarks

Our data also have fundamental implications for immune monitoring in general. Measuring serum antibodies only, as it is commonly done in clinical trials and in the clinic, provides information only on the abundance and efficacy of the “first wall” of antibody-mediated defense. A better understanding of the “second wall” of defense, encompassing the B_mem potential against both homo- and heterotypic viruses, in contrast, requires direct assessment of the B_mem cells themselves. In support of this notion, we found that elevated frequencies of antigen-reactive IgG⁺ B_mem cells is a more sensitive measure for revealing past SARS-CoV-2 virus exposure than antibody positivity (see Figs. 2C and 3C, and manuscript in preparation). Towards the practical end, we show the feasibility of high-throughput assessment of antigen-reactive B_mem cells enabling its inclusion into the routine immune monitoring portfolio.

Acknowledgments: We thank Magdalena Tary-Lehmann, Alexey Y. Karulin and Diana Roen for their valuable discussions and comments on the manuscript. We thank Tibor Baki and Victoria Gaidaenko from CTL, and Melissa Sebok, Malachi Wickman and Jennifer Penfold from American Red Cross, for their help in acquiring PBMC from SARS-CoV-2 infected donors. This study was conducted in the Research and Development department of CTL and was fully funded from the research budget of CTL. This research was performed in fulfillment of the requirements for obtaining the degree “Dr. med. dent.” by Carla Wolf at Friedrich-Alexander University (FAU), Erlangen-Nürnberg, Germany.

Author Contributions: C.W., S.K., (Sebastian Köppert), N.B., and G.A.K. conceived, designed and performed all laboratory experiments; C.W., S.K., (Sebastian Köppert) and G.A.K. performed all data analysis and generated the figures. G.A.K., P.V.L., and SK (Stefanie Küerten) supervised data analysis and contributed to the generation of figures. C.W., G.A.K. and P.V.L. wrote the original draft of the manuscript. All authors edited and reviewed the manuscript and agreed to the version submitted for publication.

Conflicts of Interest: P.V.L. is Founder, President and CEO of CTL, a company that specializes in immune monitoring by ELISPOT. G.A.K. and N.B are employees of CTL. C.W., S.K. (Sebastian Köppert) and S.K. (Stefanie Küerten) declare no conflict of interest.

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20220624Wolfetal.2022SupplementaryMaterialsFINAL.pdf
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Antibody levels poorly reflect on the frequency of memory B cells generated following SARS-CoV-2, seasonal influenza, or EBV infection

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Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Human Subjects

2.2. Polyclonal B Cell Stimulation

2.3 Recombinant Proteins

2.4. B Cell ImmunoSpot® Assays

2.5. ImmunoSpot® Image Acquisition and SFU Counting

2.6 Bivariate Visualization of FluoroSpots

2.7. ELISA Assays

2.6. Statistical Methods

3. Results And Discussion

3.1. Establishing unambiguous frequencies of antigen-reactive B cells in PBMC

3.2. Equal overall assay performance of pre- and post-COVID-19 PBMC

3.3. Exquisite specificity of SARS-CoV-2 antigen-reactive B cell ImmunoSpot® assays

3.4. Discordance between SARS-CoV-2 antigen-reactive memory B cell frequencies and antibody levels

3.5. Discordance between influenza virus and EBV antigen-reactive memory B cell frequencies and antibody levels

3.6 High reproducibility of total and antigen-reactive IgG⁺ ASC frequency measurements

3.7. Discussion of the mechanism underlying discordance and implications

3.8. Concluding remarks

Declarations

References

Supplementary Files

Status:

Version 1

Antibody levels poorly reflect on the frequency of memory B cells generated following SARS-CoV-2, seasonal influenza, or EBV infection

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Human Subjects

2.2. Polyclonal B Cell Stimulation

2.3 Recombinant Proteins

2.4. B Cell ImmunoSpot® Assays

2.5. ImmunoSpot® Image Acquisition and SFU Counting

2.6 Bivariate Visualization of FluoroSpots

2.7. ELISA Assays

2.6. Statistical Methods

3. Results And Discussion

3.1. Establishing unambiguous frequencies of antigen-reactive B cells in PBMC

3.2. Equal overall assay performance of pre- and post-COVID-19 PBMC

3.3. Exquisite specificity of SARS-CoV-2 antigen-reactive B cell ImmunoSpot® assays

3.4. Discordance between SARS-CoV-2 antigen-reactive memory B cell frequencies and antibody levels

3.5. Discordance between influenza virus and EBV antigen-reactive memory B cell frequencies and antibody levels

3.6 High reproducibility of total and antigen-reactive IgG+ ASC frequency measurements

3.7. Discussion of the mechanism underlying discordance and implications

3.8. Concluding remarks

Declarations

References

Supplementary Files

Status:

Version 1

3.6 High reproducibility of total and antigen-reactive IgG⁺ ASC frequency measurements