Anti-SARS-CoV-2 Immunoglobulin Isotypes, and Neutralization Activity Against Viral Variants, According to BNT162b2-Vaccination and Infection History

PurposeTo compare SARS-CoV-2 antigen-specific antibody production and plasma neutralizing capacity against B.1 wild-type-like strain, and Gamma/P.1 and Delta/B.1.617.2 variants-of-concern, in subjects with different Covid-19 disease and vaccination histories.MethodsAdult subjects were: 1) Unvaccinated/hospitalized for Covid-19; 2) Covid-19-recovered followed by one BNT162b2 vaccine dose; and 3) Covid-19-naïve/2-dose BNT162b2 vaccinated. Multiplex Luminex® immunoassays measured IgG, IgA, and IgM plasma levels against SARS-CoV-2 receptor-binding domain (RBD), spike-1 (S), and nucleocapsid proteins. Neutralizing activity was determined in Vero E6 cytopathic assays.ResultsMaximum anti-RBD IgG levels were similar in Covid-19‑recovered individuals 8‒10 days after single-dose vaccination and in Covid-19-naïve subjects 7 days after 2nd vaccine dosing; both groups had ≈2‑fold higher anti-RBD IgG levels than Unvaccinated/Covid-19 subjects tracked through 2 weeks post-symptom onset. Anti-S IgG expression patterns were similar to RBD within each group, but with lower signal strengths. Viral antigen-specific IgA and IgM levels were more variable than IgG patterns. Anti-nucleocapsid immunoglobulins were not detected in Covid-19-naïve subjects. Neutralizing activity against the B.1 strain, and Gamma/P.1 and Delta/B.1.617.2 variants, was highest in Covid‑19-recovered/single-dose vaccinated subjects; although neutralization against the Delta variant in this group was only 26% compared to B.1 neutralization, absolute anti-Delta titers suggested maintained protection. Neutralizing titers against the Gamma and Delta variants were 33‒77% and 26‒67%, respectively, versus neutralization against the B.1 strain (100%) in the three groups.ConclusionThese findings support SARS-CoV-2 mRNA vaccine usefulness regardless of Covid-19 history, and confirm remarkable protection provided by a single vaccine dose in people who have recovered from Covid-19.

Serological responses to SARS-CoV-2 following non-hospitalised infection: clinical and ethnodemographic features associated with the magnitude of the antibody response

ObjectiveTo determine clinical and ethnodemographic correlates of serological responses against the SARS-CoV-2 spike glycoprotein following mild-to-moderate COVID-19.DesignA retrospective cohort study of healthcare workers who had self-isolated due to COVID-19.SettingUniversity Hospitals Birmingham NHS Foundation Trust, UK (UHBFT).Participants956 healthcare workers were recruited by open invitation via UHBFT trust email and social media between 27 April 2020 and the 8 June 2020.InterventionParticipants volunteered a venous blood sample that was tested for the presence of anti-SARS-CoV-2 spike glycoprotein antibodies. Results were interpreted in the context of the symptoms of their original illness and ethnodemographic variables.ResultsUsing an assay that simultaneously measures the combined IgG, IgA and IgM response against the spike glycoprotein (IgGAM), the overall seroprevalence within this cohort was 46.2% (n=442/956). The seroprevalence of immunoglobulin isotypes was 36.3%, 18.7% and 8.1% for IgG, IgA and IgM, respectively. IgGAM identified serological responses in 40.6% (n=52/128) of symptomatic individuals who reported a negative SARS-CoV-2 PCR test. Increasing age, non-white ethnicity and obesity were independently associated with greater IgG antibody response against the spike glycoprotein. Self-reported fever and fatigue were associated with greater IgG and IgA responses against the spike glycoprotein. The combination of fever and/or cough and/or anosmia had a positive predictive value of 92.3% for seropositivity in self-isolating individuals a time when Wuhan strain SARS-CoV-2 was predominant.Conclusions and relevanceAssays employing combined antibody detection demonstrate enhanced seroepidemiological sensitivity and can detect prior viral exposure even when PCR swabs have been negative. We demonstrate an association between known ethnodemographic risk factors associated with mortality from COVID-19 and the magnitude of serological responses in mild-to-moderate disease.

B-Cell-Specific Myd88 L252P Expression Causes a Premalignant Gammopathy Resembling IgM MGUS

A highly recurrent somatic L265P mutation in the TIR domain of the signaling adapter MYD88 constitutively activates NF-κB. It occurs in nearly all human patients with Waldenström’s macroglobulinemia (WM), a B cell malignancy caused by IgM-expressing cells. Here, we introduced an inducible leucine to proline point mutation into the mouse Myd88 locus, at the orthologous position L252P. When the mutation was introduced early during B cell development, B cells developed normally. However, IgM-expressing plasma cells accumulated with age in spleen and bone, leading to more than 20-fold elevated serum IgM titers. When introduced into germinal center B cells in the context of an immunization, the Myd88L252P mutation caused prolonged persistence of antigen-specific serum IgM and elevated numbers of antigen-specific IgM plasma cells. Myd88L252P-expressing B cells switched normally, but plasma cells expressing other immunoglobulin isotypes did not increase in numbers, implying that IgM expression may be required for the observed cellular expansion. In order to test whether the Myd88L252P mutation can cause clonal expansions, we introduced it into a small fraction of CD19-positive B cells. In this scenario, five out of five mice developed monoclonal IgM serum paraproteins accompanied by an expansion of clonally related plasma cells that expressed mostly hypermutated VDJ regions. Taken together, our data suggest that the Myd88L252P mutation is sufficient to promote aberrant survival and expansion of IgM-expressing plasma cells which in turn can cause IgM monoclonal gammopathy of undetermined significance (MGUS), the premalignant condition that precedes WM.

Association between Clinical Presentation of Multiple Myeloma, Immunoglobulin Isotypes, and Cytogenetic Risk Groups

Introduction: In multiple myeloma (MM), chromosomal abnormalities (CAs) are valuable in risk stratifying patients and predicting disease free survival. While immunoglobulin isotypes have historically contributed to MM staging, more recently established classifications of CAs have allowed for a revised staging system that better predicts disease behavior. In this retrospective study, we hypothesized that CAs correlate with disease characteristics including immunoglobulin heavy chain isotype, degree of bone marrow infiltration (PC%), and end-organ damage. Methods: MM patients diagnosed between 2013 to 2019 were included in this retrospective chart review using electronic records from two distinct sources: (1) 442 patients from an independent pathology database and (2) a validation cohort composed of 110 patients from our institution. CAs were stratified by Mayo mSMART 2.0 criteria into standard, intermediate, and high-risk groups (Mikhael et al. Mayo Clin Proc 2013). End-organ damage was defined as the presence of lytic bone lesions, anemia, hypercalcemia, or renal failure on clinical presentation. Within each cohort, associations between categorical variables were made using the chi-squared test or Fisher's exact test, as deemed appropriate. The Mann-Whitney test was used to compare between groups for continuous variables. A result was considered statistically significant at the p<0.05 level of significance. All analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC). Results: 552 MM patients were included in the study. Multi-variate analysis revealed that del(13q14), dup(1q21), t(4;14), trisomy (11q13), del(16q23), and hypodiploidy were associated with a significantly higher PC%, whereas kappa light chain only (LCO) disease was associated with a lower PC%. Higher median PC% was found when comparing the intermediate to standard CA risk group. IgA isotype was associated with intermediate risk CAs including del(13q) and standard risk CAs including t(11;14), while IgG isotype was associated with dup(1q21), and kappa LCO disease correlated with a higher rate of higher risk CAs including deletion of p53 at 17p13 and dup(1q21). With regard to clinical presentation, lytic lesions were more frequent in patients with normal cytogenetics and trisomy 11 and less frequent in IgA isotype, whereas the presence of anemia on presentation correlated with IgA isotype. Renal failure was associated with MAF translocations including t(14;16), a high-risk CA. Conclusions: We demonstrate that a relationship exists between specific CAs, immunoglobulin isotypes, and clinical presentations in MM. Our data indicate that IgA isotype is significantly associated with intermediate-risk cytogenetics including del(13q) and anemia on presentation, and that light chain disease and renal failure correlate with high risk CAs including del(17p13). These associations between biological and clinical features further support the concept of divergent cytogenetic evolution in MM as being an underlying factor leading to distinctive disease presentations. Disclosures Braunstein: Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Epizyme: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Revisiting the Impact of Immunoglobulin Isotypes in Multiple Myeloma

Background Multiple Myeloma (MM) is a malignancy of terminally differentiated B lymphocytes (plasma cells, PCs) characterized by secretion of immunoglobulins and/or light chains (LCs). Association of immunoglobulin isotypes with survival in the context of contemporary therapies and accounting for modern prognostic factors has not been determined. Methods We utilized the Flatiron Health Electronic Health Record (EHR)-derived de-identified database to source patients (pts) with newly diagnosed MM from 01/2011 to 02/2020 with documented isotype data as part of the initial diagnostic work up. This nationwide database comprises de-identified, longitudinal patient-level demographic, clinical, and outcomes data curated via technology enabled abstraction. We used self-reported race and ethnicity and constructed a composite race/ethnicity variable as used by the National Cancer Institute. Similarly, we defined baseline labs (hemoglobin, serum creatinine, calcium, LDH) as those available within 90 days of diagnosis. We compared baseline characteristics using appropriate bivariate methods. Finally, we used Kaplan-Meier methods and Cox proportional hazard regression models to compare overall survival (OS), from the date of diagnosis, among the different isotypes (IgA, IgD, IgM, Light Chain (LC), others) with IgG MM after adjusting for known prognostic variables such as FISH abnormalities, ISS, renal function, age, sex, race/ethnicity, LDH, ECOG performance status, and treatment. Results We identified 8468 patients in the database who met the inclusion criteria. Patient with IgA MM (N=1688) were more likely to have ISS-III (IgA 21% vs. IgG 16%, p<0.001), anemia (IgA 41% vs. IgG 35%, p<0.001) and t(4;14) (IgA 8% vs. IgG 4%, p<0.001) than patients with IgG MM (N=4858). LC MM (N=1748) have more renal dysfunction (LC 21% vs. IgG 11%, p<0.001) and t(11;14) (LC 17% vs. IgG 8%, p<0.001). Patients with IgD MM (N=44) were younger, more likely to be male and non-Hispanic White, have ISS-III, high LDH, anemia and renal dysfunction and t(11;14). Patients with IgM MM (N=84) had higher incidence of hypercalcemia and lower proportion of patients with high LDH (Table). Across all groups 3106 (36.7%) patients received therapy containing a proteasome inhibitor (PI) and an immunomodulatory agent based triplet (IMiD), while 1458 (17.2%) received IMiD-based doublet, 2284 (27.0%) PI-based doublet, and 2109 (24.9%) received hematopoietic cell transplantation. Patients with IgA (mOS 4.7 vs 5.6 years, p<0.001) and LC MM (4.8 vs. IgG 5.6 years, p<0.001) patients have inferior OS (Figure). The adverse prognostic impact of IgA (HR 1.2, 95% CI 1.1-1.3, p<0.001) and LC isotypes (HR 1.2, 95% CI 1.1-1.3, p<0.001) on OS persisted even after adjustment for FISH abnormalities, ISS stage, renal function, age, sex, race/ethnicity, ECOG performance status, LDH, and treatment. Conclusion Using a contemporary "real world" dataset, we illustrate the distinct clinical features of MM with different immunoglobulin isotypes. Our findings suggest that IgA and LC MM are associated with poor survival suggestive of their unique biology beyond presence of high-risk FISH abnormalities and other adverse prognostic factors. Figure Disclosures Costa: Sanofi: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy; Genentech: Consultancy; BMS: Consultancy, Honoraria; Celgene: Consultancy, Honoraria.

Genetic timestamping of plasma cells in vivo reveals tissue-specific homeostatic population turnover

Plasma cells (PCs) are essential for protection from infection, and at the origin of incurable cancers. Current studies do not circumvent the limitations of removing PCs from their microenvironment and confound formation and maintenance. Also, the investigation of PC population dynamics has mostly relied on nucleotide analog incorporation that does not label quiescent cells, a property of most PCs. The main impediment is the lack of tools to perform specific genetic manipulation in vivo. Here we characterize a genetic tool (JchaincreERT2) in the mouse that permits first-ever specific genetic manipulation in PCs in vivo, across immunoglobulin isotypes. Using this tool, we found that splenic and bone marrow PC numbers remained constant over-time with the decay in genetically labeled PCs being compensated by unlabeled PCs, supporting homeostatic population turnover in these tissues. The JchaincreERT2 tool paves the way for an in-depth mechanistic understanding of PC biology and pathology in vivo, in their microenvironment.

FLOW CYTOMETRY MULTIPLEXED METHOD FOR THE DETECTION OF NEUTRALIZING HUMAN ANTIBODIES TO THE NATIVE SARS-CoV-2 SPIKE PROTEIN

A correct identification of seropositive individuals for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID-19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer-friendly non-adherent Jurkat T cell line that stably expresses the full-length native spike S protein of SARS-CoV-2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self-cleaving sequence, allowing to accurately quantify the presence of anti-S immunoglobulins by calculating a ratio of the mean fluorescence intensities obtained by double- staining with the sera and a monoclonal antibody specific for EGFR. We show that the method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. It can also be employed to detect neutralizing activity in the sera of individuals. Finally, the method can be used in a multiplexed format to simultaneously measure all anti-S human immunoglobulin isotypes in blood and mucosal fluids including total saliva.

Immunoglobulin T genes in Neopterygii

In teleost fishes there are three immunoglobulin isotypes named immunoglobulin M (IgM), D (IgD) and T (IgT). IgT has been the last to be described and is considered a teleosts-fish specific isotype. From the recent availability of genome sequences of fishes, an in-depth analysis of Actinopterygii immunoglobulin heavy chain genes was undertaken. With the aid of a bioinformatics pipeline, a machine learning software, CHfinder, was developed that identifies the coding exons of the CH domains of fish immunoglobulins. Using this pipeline, a high number of such sequences were obtained from teleosts and holostean fishes. IgT was found in teleost and holostean fishes that had not been previously described. A phylogenetic analysis reveals that IgT CH1 exons are similar to the IgM CH1. This analysis also demonstrates that the other three domains (CH2, CH3 and CH4) were not generated by recent duplication processes of IgM in Actinopterygii, indicating it is an immunoglobulin with an earlier origin.