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Respiratory syncytial virus (RSV), human metapneumovirus (HMPV), and human parainfluenza virus types one (HPIV1) and three (HPIV3) can cause severe disease and death in immunocompromised patients, the elderly, and those with underlying lung disease. A protective monoclonal antibody exists for RSV, but clinical use is limited to high-risk infant populations. Hence, therapeutic options for these viruses in vulnerable patient populations are currently limited. Here, we present the discovery, in vitro characterization, and in vivo efficacy testing of two cross-neutralizing monoclonal antibodies, one targeting both HPIV3 and HPIV1 and the other targeting both RSV and HMPV. The 3 × 1 antibody is capable of targeting multiple parainfluenza viruses; the MxR antibody shares features with other previously reported monoclonal antibodies that are capable of neutralizing both RSV and HMPV. We obtained structures using cryo-electron microscopy of these antibodies in complex with their antigens at 3.62 Å resolution for 3 × 1 bound to HPIV3 and at 2.24 Å for MxR bound to RSV, providing a structural basis for in vitro binding and neutralization. Together, a cocktail of 3 × 1 and MxR could have clinical utility in providing broad protection against four of the respiratory viruses that cause significant morbidity and mortality in at-risk individuals. Immunocompromised patients are vulnerable to respiratory viral infections. Here, the authors characterize cross-neutralizing antibodies to respiratory syncytial virus, human metapneumovirus, and human parainfluenza viruses and show effective protection in male hamsters.

Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response and by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses .

Characterization of the cellular participants in tissue immune responses is crucial to understanding infection, cancer, autoimmunity, allergy, graft rejection and other immunological processes. Previous reports indicate that leukocytes in lung vasculature fail to be completely removed by perfusion. Several studies suggest that intravascular staining may discriminate between tissue-localized and blood-borne cells in the mouse lung. Here we outline a protocol for the validation and use of intravascular staining to define innate and adaptive immune cells in mice. We demonstrate application of this protocol to leukocyte analyses in many tissues and we describe its use in the contexts of lymphocytic choriomeningitis virus and Mycobacterium tuberculosis infections or solid tumors. Intravascular staining and organ isolation usually takes 5–30 min per mouse, with additional time required for any subsequent leukocyte isolation, staining and analysis. In summary, this simple protocol should help enable interpretable analyses of tissue immune responses.

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.

BACKGROUNDLittle is known about pathogen-specific humoral immunity after chimeric antigen receptor-modified T (CAR-T) cell therapy for B cell malignancies.METHODSWe conducted a prospective cross-sectional study of CD19-targeted or B cell maturation antigen-targeted (BCMA-targeted) CAR-T cell therapy recipients at least 6 months posttreatment and in remission. We measured pathogen-specific IgG against 12 vaccine-preventable infections and the number of viral and bacterial epitopes to which IgG was detected (\"epitope hits\") using a serological profiling assay. The primary outcome was the proportion of participants with IgG levels above a threshold correlated with seroprotection for vaccine-preventable infections.RESULTSWe enrolled 65 children and adults a median of 20 months after CD19- (n = 54) or BCMA- (n = 11) CAR-T cell therapy. Among 30 adults without IgG replacement therapy (IGRT) in the prior 16 weeks, 27 (90%) had hypogammaglobulinemia. These individuals had seroprotection to a median of 67% (IQR, 59%-73%) of tested infections. Proportions of participants with seroprotection per pathogen were comparable to population-based studies, but most individuals lacked seroprotection to specific pathogens. Compared with CD19-CAR-T cell recipients, BCMA-CAR-T cell recipients were half as likely to have seroprotection (prevalence ratio, 0.47; 95% CI, 0.18-1.25) and had fewer pathogen-specific epitope hits (mean difference, -90 epitope hits; 95% CI, -157 to -22).CONCLUSIONSeroprotection for vaccine-preventable infections in adult CD19-CAR-T cell recipients was comparable to the general population. BCMA-CAR-T cell recipients had fewer pathogen-specific antibodies. Deficits in both groups support the need for vaccine and immunoglobulin replacement therapy studies.FUNDINGSwiss National Science Foundation (Early Postdoc Mobility grant P2BSP3_188162), NIH/National Cancer Institute (NIH/NCI) (U01CA247548 and P01CA018029), NIH/NCI Cancer Center Support Grants (P30CA0087-48 and P30CA015704-44), American Society for Transplantation and Cellular Therapy, and Juno Therapeutics/BMS.

Recipients of chimeric antigen receptor-modified T (CAR-T) cell therapies for B cell malignancies have profound and prolonged immunodeficiencies and are at risk for serious infections, including respiratory virus infections. Vaccination may be important for infection prevention, but there are limited data on vaccine immunogenicity in this population. We conducted a prospective observational study of the humoral immunogenicity of commercially available 2019–2020 inactivated influenza vaccines in adults immediately prior to or while in durable remission after CD19-, CD20-, or B cell maturation antigen-targeted CAR-T-cell therapy, as well as controls. We tested for antibodies to all four vaccine strains using neutralization and hemagglutination inhibition (HAI) assays. Antibody responses were defined as at least fourfold titer increases from baseline. Seroprotection was defined as a HAI titer ≥40. Enrolled CAR-T-cell recipients were vaccinated 14–29 days prior to (n=5) or 13–57 months following therapy (n=13), and the majority had hypogammaglobulinemia and cellular immunodeficiencies prevaccination. Eight non-immunocompromised adults served as controls. Antibody responses to ≥1 vaccine strain occurred in 2 (40%) individuals before CAR-T-cell therapy and in 4 (31%) individuals vaccinated after CAR-T-cell therapy. An additional 1 (20%) and 6 (46%) individuals had at least twofold increases, respectively. One individual vaccinated prior to CAR-T-cell therapy maintained a response for >3 months following therapy. Across all tested vaccine strains, seroprotection was less frequent in CAR-T-cell recipients than in controls. There was evidence of immunogenicity even among individuals with low immunoglobulin, CD19+ B cell, and CD4+ T-cell counts. These data support consideration for vaccination before and after CAR-T-cell therapy for influenza and other relevant pathogens such as SARS-CoV-2, irrespective of hypogammaglobulinemia or B cell aplasia. However, relatively impaired humoral vaccine immunogenicity indicates the need for additional infection-prevention strategies. Larger studies are needed to refine our understanding of potential correlates of vaccine immunogenicity, and durability of immune responses, in CAR-T-cell therapy recipients.

Monoclonal antibodies specific for foreign antigens, auto-antigens, allogeneic antigens and tumour neo-antigens in the context of major histocompatibility complex II (MHCII) are highly desirable as novel immunotherapeutics. However, there is no standard protocol for the efficient generation of monoclonal antibodies that recognize peptide in the context of MHCII, and only a limited number of such reagents exist. In this report, we describe an approach for the generation and screening of monoclonal antibodies specific for peptide bound to MHCII. This approach exploits the use of recombinant peptide:MHC monomers as immunogens, and subsequently relies on multimers to pre-screen and magnetically enrich the responding antigen-specific B cells before fusion and validation, thus saving significant time and reagents. Using this method, we have generated two antibodies enabling us to interrogate antigen presentation and T-cell activation. This methodology sets the standard to generate monoclonal antibodies against the peptide–MHCII complexes. Generating antibodies specific for the peptide–MHCII complexes has been challenging, with only a handful made to date. Here, the authors develop a more efficient approach to generate these antibodies, and demonstrate their potential in research and therapeutic applications.

Background/Objectives: Vaccines targeting melanoma antigens can elicit CD8+ T cell responses, but a growing body of work suggests CD4+ T cells also play a role in tumor control. Induction of CD4+ cells may also support B cells in producing tumor antigen-specific antibodies (Abs). We investigated Abs induced by vaccination with a cocktail of six class II MHC-restricted melanoma peptides (6MHP) and the effect of adjuvant type on Ab isotypes. We hypothesized that the vaccines would induce Abs that respond to different epitopes on individual peptides and that IgG isotype distribution varies with different vaccine adjuvants. Methods: Sera from patients who received a 6MHP vaccine were evaluated with enzyme-linked immunosorbent assays to map epitopes for polyclonal Ab responses to synthetic melanoma peptides. IgG isotypes of Ab responses to 6MHP were assessed in patients who received one of four adjuvants (Incomplete Freund’s Adjuvant (IFA) alone, IFA + polyICLC, IFA + systemic metronomic cyclophosphamide (mCy), or IFA + polyICLC + systemic mCy) to characterize IgG isotype distribution. Results: Epitope mapping revealed that at least 50% of patients had responses to two or more epitopes on the same peptide, suggesting polyclonal Ab responses. Serum evaluation for IgG isotypes showed predominant induction of IgG1 and IgG3. Mean total IgG was highest when IFA and polyICLC were used in combination. Patients who received TLR3 agonist polyICLC had significantly higher concentrations of total IgG, IgG1, and IgG3 compared to patients who did not receive polyICLC. Conclusions: Vaccine-induced Abs may respond to multiple epitopes within the same peptide, warranting further studies into their ability to facilitate antigen uptake and presentation through the formation of large immune complexes. The findings also show that adding polyICLC to IFA can significantly enhance Ab responses. Collectively, this work underscores the immunologic potential of peptide-induced Abs and the importance of adjuvant selection in cancer vaccine design.

ANCAs are diagnostic and pathogenic in ANCA-associated vasculitis however ANCAs can persist or reappear in clinical remission. It is a known phenomenon that naturally occurring autoantibodies, including ANCAs, are also detectable in healthy individuals. That autoantibodies do not directly correlate with clinical disease suggests that characteristics of the autoantibody response, rather than its mere presence, may determine pathogenicity. We aimed to define immunogenomic and epitope-level differences between naturally occurring and disease-associated MPO-ANCA. Patients with MPO-ANCA vasculitis (active disease and long-term remission off therapy) and healthy individuals were enrolled at a single center. Peripheral blood mononuclear cells were screened using highly sensitive methods to isolate MPO specific B cells from healthy individuals and patients with MPO-ANCA. Sorted cells were expanded in vitro with antigen stimulation, and supernatants were tested for MPO-ANCA IgG. Bulk B cell receptor (BCR) sequencing of heavy and light chains was performed on autoantigen-specific and control B cells. Serum IgG from the same cohort was used to map MPO epitopes using a yeast surface display library of overlapping MPO-derived peptides. We found that autoantigen-specific B cells were present at similar frequencies in patients with active disease, patients in long-term remission off therapy, and healthy individuals. However, only MPO specific B cells from MPO-ANCA patients produced high levels of MPO-ANCA in vitro, whereas MPO specific B cells from healthy individuals did not. MPO specific B cells from patients exhibited shared immunogenomic features such as reduced clonotype diversity and clonal dominance compared with non-MPO B cells, and there was greater overlap in CDR3 sequences. Epitope mapping revealed that healthy individuals had naturally occurring MPO-reactive IgG, however patient IgG conferred distinct MPO epitope signatures. Our findings demonstrate that naturally occurring and disease-associated MPO-ANCA differ in both BCR repertoire features and epitope specificity. These data support the concept that immunological remission in MPO-ANCA vasculitis cannot be defined solely by the presence or absence of ANCAs, but depends on qualitative differences in the autoantibody response, including BCR sequence and epitope targeting.