Boosting corrects a memory B cell defect in SARS-CoV-2 mRNA-vaccinated patients with inflammatory bowel disease.

Immunosuppressed patients with inflammatory bowel disease (IBD) generate lower amounts of SARS-CoV-2 spike antibodies after mRNA vaccination than healthy controls. We assessed SARS-CoV-2 spike S1 receptor binding domain-specific (S1-RBD-specific) B lymphocytes to identify the underlying cellular defects. Patients with IBD produced fewer anti-S1-RBD antibody-secreting B cells than controls after the first mRNA vaccination and lower amounts of total and neutralizing antibodies after the second. S1-RBD-specific memory B cells were generated to the same degree in IBD and control groups and were numerically stable for 5 months. However, the memory B cells in patients with IBD had a lower S1-RBD-binding capacity than those in controls, which is indicative of a defect in antibody affinity maturation. Administration of a third shot to patients with IBD elevated serum antibodies and generated memory B cells with a normal antigen-binding capacity. These results show that patients with IBD have defects in the formation of antibody-secreting B cells and affinity-matured memory B cells that are corrected by a third vaccination.

High-affinity memory B cells induced by SARS-CoV-2 infection produce more plasmablasts and atypical memory B cells than those primed by mRNA vaccines.

Although both infections and vaccines induce memory B cell (MBC) populations that participate in secondary immune responses, the MBCs generated in each case can differ. Here, we compare SARS-CoV-2 spike receptor binding domain (S1-RBD)-specific primary MBCs that form in response to infection or a single mRNA vaccination. Both primary MBC populations have similar frequencies in the blood and respond to a second S1-RBD exposure by rapidly producing plasmablasts with an abundant immunoglobulin (Ig)A+ subset and secondary MBCs that are mostly IgG+ and cross-react with the B.1.351 variant. However, infection-induced primary MBCs have better antigen-binding capacity and generate more plasmablasts and secondary MBCs of the classical and atypical subsets than do vaccine-induced primary MBCs. Our results suggest that infection-induced primary MBCs have undergone more affinity maturation than vaccine-induced primary MBCs and produce more robust secondary responses.

Initial determination of COVID-19 seroprevalence among outpatients and healthcare workers in Minnesota using a novel SARS-CoV-2 total antibody ELISA.

OBJECTIVES: To avoid the significant risks posed by the use of COVID-19 serology tests with supply chain constraints or poor performance characteristics, we developed an in-house SARS-CoV-2 total antibody test. Our test was compared with three commercial methods, and was used to determine COVID-19 seroprevalence among healthcare workers and outpatients in Minnesota. METHODS: Seventy-nine plasma and serum samples from 50 patients 4-69 days after symptom onset who tested positive by a SARS-CoV-2 PCR method using a nasopharyngeal (NP) swab were used to evaluate our test's clinical performance. Seropositive samples were analyzed for IgG titers in a follow-up assay. Thirty plasma and serum from 12 patients who tested negative by a SARS-CoV-2 PCR method using a nasopharyngeal (NP) swab and 210 negative pre-pandemic serum samples were also analyzed. Among samples from patients > 14 days after symptom onset, the assay had 100% clinical sensitivity and 100% clinical specificity, 100% positive predictive value and 100% negative predictive value. Analytical specificity was 99.8%, indicating minimal cross-reactivity. A screening study was conducted to ascertain COVID-19 seroprevalence among healthcare workers and outpatients in Minnesota. RESULTS: Analysis of serum collected between April 13 and May 21, 2020 indicated a COVID-19 seroprevalence of 2.96% among 1,282 healthcare workers and 4.46% among 2,379 outpatients. CONCLUSIONS: Our in-house SARS-CoV-2 total antibody test can be used to conduct reliable epidemiological studies to inform public health decisions during the COVID-19 pandemic.

SARS-CoV-2 neutralization and serology testing of COVID-19 convalescent plasma from donors with nonsevere disease.

BACKGROUND: The transfer of passive immunity with convalescent plasma is a promising strategy for treatment and prevention of COVID-19, but donors with a history of nonsevere disease are serologically heterogenous. The relationship between SARS-Cov-2 antigen-binding activity and neutralization activity in this population of donors has not been defined. STUDY DESIGN AND METHODS: Convalescent plasma units from 47 individuals with a history of nonsevere COVID-19 were assessed for antigen-binding activity of using three clinical diagnostic serology assays (Beckman, DiaSorin, and Roche) with different SARS-CoV-2 targets. These results were compared with functional neutralization activity using a fluorescent reporter strain of SARS-CoV-2 in a microwell assay. RESULTS: Positive correlations of varying strength (Spearman r = 0.37-0.52) between antigen binding and viral neutralization were identified. Donors age 48 to 75 years had the highest neutralization activity. Units in the highest tertile of binding activity for each assay were enriched (75%-82%) for those with the highest levels of neutralization. CONCLUSION: The strength of the relationship between antigen-binding activity and neutralization varies depending on the clinical assay used. Units in the highest tertile of binding activity for each assay are predominantly comprised of those with the greatest neutralization activity.

SARS-CoV-2 neutralization and serology testing of COVID-19 convalescent plasma from donors with non-severe disease.

We determined the antigen binding activity of convalescent plasma units from 47 individuals with a history of non-severe COVID-19 using three clinical diagnostic serology assays (Beckman, DiaSorin, and Roche) with different SARS-CoV-2 targets. We compared these results with functional neutralization activity using a fluorescent reporter strain of SARS-CoV-2 in a microwell assay. This revealed positive correlations of varying strength (Spearman r = 0.37-0.52) between binding and neutralization. Donors age 48-75 had the highest neutralization activity. Units in the highest tertile of binding activity for each assay were enriched (75-82%) for those with the highest levels of neutralization.

Polymicrobial Sepsis Chronic Immunoparalysis Is Defined by Diminished Ag-Specific T Cell-Dependent B Cell Responses.

Immunosuppression is one hallmark of sepsis, decreasing the host response to the primary septic pathogens and/or secondary nosocomial infections. CD4 T cells and B cells are among the array of immune cells that experience reductions in number and function during sepsis. "Help" from follicular helper (Tfh) CD4 T cells to B cells is needed for productive and protective humoral immunity, but there is a paucity of data defining the effect of sepsis on a primary CD4 T cell-dependent B cell response. Using the cecal ligation and puncture (CLP) mouse model of sepsis induction, we observed reduced antibody production in mice challenged with influenza A virus or TNP-KLH in alum early (2 days) and late (30 days) after CLP surgery compared to mice subjected to sham surgery. To better understand how these CD4 T cell-dependent B cell responses were altered by a septic event, we immunized mice with a Complete Freund's Adjuvant emulsion containing the MHC II-restricted peptide 2W1S56-68 coupled to the fluorochrome phycoerythrin (PE). Immunization with 2W1S-PE/CFA results in T cell-dependent B cell activation, giving us the ability to track defined populations of antigen-specific CD4 T cells and B cells responding to the same immunogen in the same mouse. Compared to sham mice, differentiation and class switching in PE-specific B cells were blunted in mice subjected to CLP surgery. Similarly, mice subjected to CLP had reduced expansion of 2W1S-specific T cells and Tfh differentiation after immunization. Our data suggest CLP-induced sepsis impacts humoral immunity by affecting the number and function of both antigen-specific B cells and CD4 Tfh cells, further defining the period of chronic immunoparalysis after sepsis induction.

Naive B Cells with High-Avidity Germline-Encoded Antigen Receptors Produce Persistent IgM+ and Transient IgG+ Memory B Cells.

Although immune memory often lasts for life, this is not the case for certain vaccines in some individuals. We sought a mechanism for this phenomenon by studying B cell responses to phycoerythrin (PE). PE immunization of mouse strains with Ighb immunoglobulin (Ig) variable heavy chain (VH) genes elicited affinity-matured switched Ig memory B cells that declined with time, while the comparable population from an Igha strain was numerically stable. Ighb strains had larger numbers of PE-specific naive B cells and generated smaller germinal center responses and larger numbers of IgM memory cells than the Igha strain. The properties of PE-specific B cells in Ighb mice correlated with usage of a single VH that afforded high-affinity PE binding in its germline form. These results suggest that some individuals may be genetically predisposed to generate non-canonical memory B cell responses to certain antigens because of avid antigen binding via germline-encoded VH elements.

Do Memory B Cells Form Secondary Germinal Centers? It Depends.

The memory B-cell pool in an immune individual is more heterogeneous than previously recognized. The different types of memory B cells likely play distinct roles in tuning the secondary immune response because they differ in their potential to generate plasmablasts, which secrete antibodies, or germinal center (GC) cells, which generate new and higher affinity memory cells. We propose that the production of plasmablasts or GC cells by a memory B cell is controlled by its state of differentiation and the amount and affinity of antigen-specific antibodies present in the individual in which it resides.

Humoral immunity. Apoptosis and antigen affinity limit effector cell differentiation of a single naïve B cell.

When exposed to antigens, naïve B cells differentiate into different types of effector cells: antibody-producing plasma cells, germinal center cells, or memory cells. Whether an individual naïve B cell can produce all of these different cell fates remains unclear. Using a limiting dilution approach, we found that many individual naïve B cells produced only one type of effector cell subset, whereas others produced all subsets. The capacity to differentiate into multiple subsets was a characteristic of clonal populations that divided many times and resisted apoptosis, but was independent of isotype switching. Antigen receptor affinity also influenced effector cell differentiation. These findings suggest that diverse effector cell types arise in the primary immune response as a result of heterogeneity in responses by individual naïve B cells.

Heterogeneity in the differentiation and function of memory B cells.

Vaccines that induce neutralizing antibodies have led to the eradication of small pox and have severely reduced the prevalence of many other infections. However, even the most successful vaccines do not induce protective antibodies in all individuals, and can fail to induce lifelong immunity. A key to remedying these shortcomings may lie in a better understanding of long-lived memory B cells. Recent studies have revealed novel insights into the differentiation and function of these cells, and have shown that the memory B cell pool is much more heterogeneous than previously appreciated.

A germinal center-independent pathway generates unswitched memory B cells early in the primary response.

Memory B cells can be produced from the classical germinal center (GC) pathway or a less understood GC-independent route. We used antigen-based cell enrichment to assess the relative contributions of these pathways to the polyclonal memory B cell pool. We identified a CD38(+) GL7(+) B cell precursor population that differentiated directly into IgM(+) or isotype-switched (sw) Ig(+) memory B cells in a GC-independent fashion in response to strong CD40 stimulation. Alternatively, CD38(+) GL7(+) B cell precursors had the potential to become Bcl-6(+) GC cells that then generated primarily swIg(+) memory B cells. These results demonstrate that early IgM(+) and swIg(+) memory B cells are products of a GC-independent pathway, whereas later switched Ig(+) memory B cells are products of GC cells.

Different B cell populations mediate early and late memory during an endogenous immune response.

Memory B cells formed in response to microbial antigens provide immunity to later infections; however, the inability to detect rare endogenous antigen-specific cells limits current understanding of this process. Using an antigen-based technique to enrich these cells, we found that immunization with a model protein generated B memory cells that expressed isotype-switched immunoglobulins (swIg) or retained IgM. The more numerous IgM(+) cells were longer lived than the swIg(+) cells. However, swIg(+) memory cells dominated the secondary response because of the capacity to become activated in the presence of neutralizing serum immunoglobulin. Thus, we propose that memory relies on swIg(+) cells until they disappear and serum immunoglobulin falls to a low level, in which case memory resides with durable IgM(+) reserves.

A protease-dependent mechanism for initiating T-dependent B cell responses to large particulate antigens.

Ab production is critical for antimicrobial immunity, and the initial step in this process is the binding of Ag to the BCR. It has been shown that small soluble proteins can directly access the lymph node follicles to reach naive B cells, but virus particles must be translocated into follicles via subcapsular sinus macrophages. In this article, we explore how large particulate Ags generate humoral immune responses. Ag-specific follicular B cells rapidly acquired Ag, presented peptide:MHC class II ligands, and produced T-dependent Ab responses following s.c. injection of 1-mum, Ag-linked microspheres, despite the microspheres being confined to the subcapsular sinus. The mechanism of Ag acquisition did not require dendritic cells, subcapsular sinus macrophages, or B cell movement to the subcapsular sinus. Rather, B cell Ag acquisition was protease-dependent, suggesting that some protein Ags are cleaved from the surface of particles to directly initiate humoral immune responses.

The humoral immune response is initiated in lymph nodes by B cells that acquire soluble antigen directly in the follicles.

The initial step in a humoral immune response involves the acquisition of antigens by B cells via surface immunoglobulin. Surprisingly, anatomic studies indicate that lymph-borne proteins do not have access to the follicles where naive B cells reside. Thus, it is unclear how B cells acquire antigens that drain to lymph nodes. By tracking a fluorescent antigen and a peptide:MHC II complex derived from it, we show that antigen-specific B cells residing in the follicles acquire antigen within minutes of injection, first in the region closest to the subcapsular sinus where lymph enters the lymph node. Antigen acquisition, presentation, and subsequent T cell-dependent activation did not require B cell migration through the T cell area or exposure to dendritic cells. These results indicate that the humoral response is initiated as soluble antigens diffuse directly from lymph in the subcapsular sinus to be acquired by antigen-specific B cells in the underlying follicles.

In vivo assessment of the relative contributions of deletion, anergy, and editing to B cell self-tolerance.

In normal B cell development, a large percentage of newly formed cells bear receptors with high levels of self-reactivity that must be tolerized before entry into the mature B cell pool. We followed the fate of self-reactive B cells expressing high affinity anti-hen egg lysozyme (HEL) Ag receptors exposed in vivo to membrane HEL in a setting in which the anti-HEL L chain was "knocked-in" at the endogenous L chain locus. These mice demonstrated extensive and efficient L chain receptor editing responses and had B cell numbers comparable to those found in animals lacking membrane Ag. BrdU labeling indicated that the time required for editing in response to membrane HEL was approximately 6 h. In mice transgenic for soluble HEL, anti-HEL B cells capable of editing showed evidence for both editing and anergy. These data identify receptor editing as a major physiologic mechanism by which highly self-reactive B cells are tolerized to membrane and soluble self-Ags.

Visualizing the first 50 hr of the primary immune response to a soluble antigen.

Recently, static and dynamic imaging methods have produced the first glimpses of the interactions between antigen-specific T cells and peptide-MHC-bearing antigen-presenting cells in the lymph nodes. Using data from these experiments, we produced a numerically, spatially, and temporally scaled simulation of the first 50 hr of the primary T cell-dependent immune response. The simulation highlights how lymph node structure facilitates antigen presentation to rare, naive, antigen-specific CD4+ T cells.

Visualization of the genesis and fate of isotype-switched B cells during a primary immune response.

The life history of isotype-switched B cells is unclear, in part, because of an inability to detect rare antigen-specific B cells at early times during the immune response. To address this issue, a small population of B cells carrying targeted antibody transgenes capable of class switching was monitored in immunized mice. After contacting helper T cells, the first switched B cells appeared in follicles rather than in the red pulp, as was expected. Later, some of the switched B cells transiently occupied the red pulp and marginal zone, whereas others persisted in germinal centers (GCs). Antigen-experienced IgM B cells were rarely found in GCs, indicating that these cells switched rapidly after entering GCs or did not persist in this environment.