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Effect of B cell depletion on natural IgM mediated immunity against S. pneumoniae. Flow cytometry analysis of splenocytes recovered from C57B/6 mice 7 days after IP administration of 100ug of aCD20 antibody (six mice/group). (A) Percentage of persisting natural IgM secreting cells (B1a, CD5+CD23-) and total B cells (CD19+CD3-) compared to undepleted controls. (B) A representative flow cytometry plot of the relative level of IgM expression in splenic B cells from aCD20 treated (solid line) and untreated controls (dotted line). Unstained sample data are also shown (gray filling). (C) IgM expression level (Mean fluorescence intensity - MFI) on B1a cells (CD5+CD23-) in the depleted (white bar) versus undepleted mice (black bar). (D) A representative flow cytometry plot of IgM binding to S. pneumoniae 6B strain incubated in serum from undepleted (dotted line) and B cell depleted (solid line) mice; unstained samples (gray filling). (E) Mean MFI of IgM binding to S. pneumoniae 6B strain in serum from B cell depleted (white column) or undepleted (black columns) C57B/6 mice. (F, G) Effects of B cell depletion of immune naïve C57B/6 mice on susceptibility to S. pneumoniae pneumonia. Mice were inoculated intranasally with 2 x 10⁶ CFU S. pneumoniae 6B (F) and D39 strain (G) CFU in 50 ul PBS under deep anaesthesia and CFU were measured at 24 h by plating serial dilutions of blood and lungs. For all graphs, bars represent the mean values for each group, error bars indicate standard deviations and Mann Whitney U test was used for statistical analysis (*P < 0.05, ns, not significant).
Source publication
The anti-CD20 antibody Rituximab to deplete CD20+ B cells is an effective treatment for rheumatoid arthritis and B cell malignancies, but is associated with an increased incidence of respiratory infections. Using mouse models we have investigated the consequences of B cell depletion on natural and acquired humoral immunity to Streptococcus pneumoni...
Citations
... Prebleed sera showed minimal reactivity to all 50 of these antigens. This protein array was previously used to probe antibody responses to Spn proteins in mice (12,25), and our data show reactivity to a considerably larger number of Spn proteins for Gamma-PN-immunized rabbits compared to prior studies. Interestingly, of the 50 top antigens, 11 fell below the threshold of detection when Gamma-PN was adjuvanted, supporting the notion of impaired antibody reactivity. ...
ABSTRACT Streptococcus pneumoniae (Spn) remains a major cause of global mortality, with extensive antigenic diversity between capsular serotypes that poses an ongoing challenge for vaccine development. Widespread use of pneumococcal conjugate vaccines (PCVs) targeting Spn capsules has greatly reduced infections by vaccine-included serotypes but has led to increased infections by nonincluded serotypes. To date, high cost of PCVs has also limited their usefulness in low-income regions where disease burdens are highest. To overcome these limitations, serotype-independent vaccines are being actively researched. We have developed a whole-cell gamma-irradiated Spn vaccine (termed Gamma-PN) providing serotype-independent protection. We demonstrate that Gamma-PN immunization of mice or rabbits via the clinically relevant intramuscular route induces protein-specific antibodies able to bind numerous nonvaccine encapsulated serotypes, which mediate opsonophagocytic killing and protection against lethal challenges. Gamma-PN induced comparable or superior opsonophagocytic killing assay (OPKA) responses in rabbits to the licensed Prevnar 13 vaccine (PCV13) for vaccine-included serotypes, and a superior response to nonincluded serotypes, including emergent 22F and 35B. Additionally, despite a lower observed reactogenicity, administration of Gamma-PN without adjuvant resulted in higher OPKA responses and improved protection compared to adjuvanted Gamma-PN. To our knowledge, this has not been demonstrated previously for a whole-inactivated Spn vaccine. Eliminating the requirement for adjuvant comes with numerous benefits for clinical applications of this vaccine and poses interesting questions for the inclusion of adjuvant in similar vaccines in development. IMPORTANCE The target pathogen of this study, Streptococcus pneumoniae, kills over 300,000 children
... The early adaptive immune response delays the spread of pneumococci from the brain to the systemic circulation and shapes the immune response [15].γδ T cell accumulates, and activates and supports pneumococcal clearance during S. pneumoniae infection [16,17]. Recent studies also showed that IgG antibodies and Th17 cells form naturally acquired adaptive immunity against pneumococci in humans, and B cells produce natural IgM antibodies against pneumococci by improving the complement-mediated systemic immunity [18,19]. ...
Streptococcus pneumoniae is a Gram-positive, human-specific commensal infectious pathogen that can lead to death worldwide, especially in children under five. A deeper understanding of the immune response in pneumococcal meningitis (PM) has high scientific importance. In this study, we performed single-cell RNA sequencing of peripheral blood mononuclear cells from PM patients and healthy individuals. Both platelets and neutrophils were found with higher proportions in the PM group compared to the control group. Of the differentially expressed genes (DEGs), the genes involved in the immune functions of neutrophils were up-regulated in many immune cells, including platelets, myeloid cells and B cells, suggesting excessive neutrophil activation; while the genes involved in antigen processing and presentation in myeloid cells and B cells were down-regulated in the PM group, which indicated adaptive immune deficiency in the PM patients. Consistently, the proportions of plasmacytoid dendritic cells and T cells were decreased. Furthermore, genes involved in cytotoxity were down-regulated in both NK cells and T cells, and T cell activation was deficient. Many DEGs were also enriched in some signaling pathways that mediate interactions between S. pneumoniae and immune cells, including the Toll-like receptor pathway, the Nucleotide oligomerization domain-like signaling pathway, and the tumor necrosis factor signaling pathway. In conclusion, our study provided a global characterization of S. pneumoniae -induced systemic immune responses, which may have implications for identification of potential targets to improve the immune response in PM.
... We have previously established a mouse model of B-cell depletion in which one dose (50 µg) of anti-CD20 antibody causes over 95% reduction in spleen, lymph node and circulating B-cell numbers. 20 In this model, total B cells numbers were fully restored after three weeks, although reconstitution of the follicular B-cell subset population was slightly impaired. To investigate whether established serological responses to vaccination are affected by subsequent B-cell depletion treatment, C57BL/6 mice received two doses of Prevnar-13 vaccine then were given one dose of B-cell depletion therapy 14 days after the second vaccination ( Figure 1a). ...
... Under these conditions, PCV was administered to animals at a timepoint when their B cell resident population was reduced to less than 5% of the original. 20 For the B-cell depletion group total serum IgG level was reduced compared to vaccinated controls non-B-cell-depleted mice but was unchanged compared to control mice total IgG ( Figure 3b). B-cell depletion just before vaccination had a profound impact on the serological responses to the PCV vaccine. ...
Objectives
Anti-CD20 monoclonal antibody therapy rapidly depletes > 95% of CD20⁺ B cells from the circulation. B-cell depletion is an effective treatment for autoimmune disease and B-cell malignancies but also increases the risk of respiratory tract infections. This effect on adaptive immunity could be countered by vaccination. We have used mouse models to investigate the effects of B-cell depletion on pneumococcal vaccination, including protection against infection and timing of vaccination in relation to B-cell depletion.
Methods
C57BL/6 female mice were B-cell depleted using anti-CD20 antibody and immunized with two doses of Prevnar-13 vaccine either before or after anti-CD20 treatment. B-cell repertoire and Streptococcus pneumoniae–specific IgG levels were measured using whole-cell ELISA and flow cytometry antibody-binding assay. Protection induced by vaccination was assessed by challenging the mice using a S. pneumoniae pneumonia model.
Results
Antibody responses to S. pneumoniae were largely preserved in mice B-cell depleted after vaccination resulting in full protection against pneumococcal infections. In contrast, mice vaccinated with Prevnar-13 while B cells were depleted (with > 90% reduction in B-cell numbers) had decreased circulating anti–S. pneumoniae IgG and IgM levels (measured using ELISA and flow cytometry antibody binding assays). However, some antibody responses were maintained, and, although vaccine-induced protection against S. pneumoniae infection was impaired, septicaemia was still prevented in 50% of challenged mice.
Conclusions
This study showed that although vaccine efficacy during periods of profound B-cell depletion was impaired some protective efficacy was preserved, suggesting that vaccination remains beneficial.
... The protein microarray contained 254 proteins selected based on conservation from an original panel of >600 S. pneumoniae strains [23,37,38]. Exons were amplified from genomic DNA (strain TIGR4) and cloned into a T7 expression vector. ...
Rationale:
Nasopharyngeal administration of live virulence-attenuated Streptococcus pneumoniae strains is a potential novel preventative strategy. One target for creating reduced virulence S. pneumoniae strains is the capsule, but loss of the capsule reduces the duration of S. pneumoniae colonisation in mice which could impair protective efficacy against subsequent infection.
Objectives:
To assess protective efficacy of nasopharyngeal administration of unencapsulated S. pneumoniae strains in murine infection models.
Methods:
Strains containing cps locus deletions combined with the S. pneumoniae virulence factors psaA (reduces colonisation) or proABC (no effect on colonisation) were constructed and their virulence phenotypes and ability to prevent recolonisation or invasive infection assessed using mouse infection models. Serological responses to colonisation were compared between strains using ELISAs, immunoblots and 254 S. pneumoniae protein antigen array.
Measurements and main results:
The ∆cps/piaA and ∆cps/proABC strains were strongly attenuated in virulence in both invasive infection models and had a reduced ability to colonise the nasopharynx. ELISAs, immunoblots and protein arrays showed colonisation with either strain stimulated weaker serological responses than the wild type strain. Mice previously colonised with these strains were protected against septicaemic pneumonia but, unlike mice colonised with the wild type strain, not against S. pneumoniae recolonisation.
Conclusions:
Colonisation with the ∆cps/piaA and ∆cps/proABC strains prevented subsequent septicaemia, but in contrast, to published data for encapsulated double mutant strains they did not prevent recolonisation with S. pneumoniae. These data suggest targeting the cps locus is a less effective option for creating live attenuated strains that prevent S. pneumoniae infections.
Purpose: Patients with rheumatoid arthritis (RA) have an increased susceptibility to infection, including those caused by Streptococcus pneumoniae. Why RA is associated with increased susceptibility to S. pneumoniae is poorly understood. This study aims to assess the effects of RA and B cell depletion therapy on naturally acquired antibody responses to 289 S. pneumoniae protein antigens using a novel protein array.
Methods: IgG responses to S. pneumoniae were characterized in serum from RA patients and disease controls (myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)) using whole-cell ELISA, a flow cytometry opsonization assay, and a S. pneumoniae protein array. For the RA patients, results were compared before and after B cell depletion therapy.
Results: Compared to a well characterized disease control group of ME/CFS patients, RA patients had reduced antibody responses to multiple S. pneumoniae protein antigens, with significant IgG recognition of approximately half the number of antigens along with reduced median strengths of these responses. Reduction in multiple array antigen-specific responses also correlated with reduced IgG opsonization of S. pneumoniae. Although B cell depletion therapy with rituximab did not reduce overall IgG recognition of S. pneumoniae in the RA group, it was associated with marked disruption of pre-existing IgG repertoire to protein antigens in individual patients.
Conclusion: These data show RA is associated with major disruption of naturally acquired adaptive immunity to S. pneumoniae, which can be assessed rapidly using a protein antigen array and is likely to contribute towards the increased incidence of pneumonia in patients with RA.
Background
CD19 targeted CAR T cell therapy (CAR-T) leads to B cell aplasia and low serum immunoglobulin levels. Long-lived CD19 negative plasma cells may persist through the therapy and generate antibodies. There is a paucity of data describing how CAR-T impacts the persistence of antibodies against vaccine-related antigens and the degree to which CAR-T recipients may respond to vaccines.
Objective
We characterized the effect of CAR-T on pneumococcal IgG titers and determine whether pneumococcal conjugate vaccine (PCV13) administered after CAR-T develops long-term humoral protection against pneumococcus.
Study Design
A retrospective chart review was performed to identify CAR-T recipients that had serum pneumococcal IgG titers drawn before (baseline) or at days+90, +180, +270, +360, or +540 after CAR-T. We then determined whether they received PCV13 vaccination at these timepoints. IgG concentration ≥1.3μg/mL was considered protective for that serotype, and patients with ≥6/11 tested vaccine specific serotypes meeting this threshold were deemed to have humoral protection against pneumococcus. Absolute pneumococcal IgG titers and the proportion of patients with humoral protection, stratified by serotype and vaccination status, were compared by paired nonparametric t-tests. Absolute counts for lymphocyte, CD4 T cell and CD19 cell and total IgG level, along with the rate of invasive pneumococcal infections were measured at these timepoints.
Results
A total of 148 CAR-T recipients with pneumococcal IgG titers measured for at least one of the defined time points were identified. At baseline, 25% (19/76) patients with evaluable pneumococcal IgG titers met the definition of humoral protection. Among 44 patients with paired pneumococcal IgG titers at baseline and day+90, absolute IgG titers of all serotypes decreased (geometric mean= 0.41 and 0.32 µg/ml, respectively; P<0.001). Thirteen patients were vaccinated following the titer blood draw at day+90 and had paired pneumococcal IgG titers at day+90 and day+180. Absolute IgG titers of all vaccine specific serotypes in these vaccinated patients decreased from day+90 to day+180 (geometric mean= 0.36 and 0.29 µg/ml, respectively; P=0.03). The proportion of patients meeting the criteria of humoral protection remained the same at day+180 despite vaccination at day+90. The results were similar among 8 patients vaccinated at day+180 as well as 7 patients consecutively vaccinated at day+90 and day+180 with corresponding pneumococcal IgG titers. When all vaccine specific pneumococcal IgG titers were pooled together by timepoint regardless of vaccination status, the proportion of patients with humoral protection decreased until day+540. Some patients developed humoral protection following vaccination at day+360, maintained seroprotective IgG titers from baseline, or developed protection after receiving intravenous immunoglobulin treatment secondary to recurrent infections.
Conclusion
Our study demonstrated that few large B cell lymphoma patients had humoral protection against pneumococcus at baseline, and existing IgG titers decreased after CAR-T. PCV13 vaccination at day+90 and/or day+180 post CAR-T did not increase humoral protection against pneumococcus. Only at day+540 was there evidence of humoral protection against pneumococcus in a modest proportion of patients. Clinical trials are needed to determine the optimal timing of vaccination, before or after CAR-T, to develop protective immunity against S. pneumoniae infections.
The pulmonary immune system plays a vital role in protecting the delicate structures of gaseous exchange against invasion from bacterial pathogens. With antimicrobial resistance becoming an increasing concern, finding novel strategies to develop vaccines against bacterial lung diseases remains a top priority. In order to do so, a continued expansion of our understanding of the pulmonary immune response is warranted. Whilst some aspects are well characterised, emerging paradigms such as the importance of innate cells and inducible immune structures in mediating protection provide avenues of potential to rethink our approach to vaccine development. In this review, we aim to provide a broad overview of both the innate and adaptive immune mechanisms in place to protect the pulmonary tissue from invading bacterial organisms. We use specific examples from several infection models and human studies to depict the varying functions of the pulmonary immune system that may be manipulated in future vaccine development. Particular emphasis has been placed on emerging themes that are less reviewed and underappreciated in vaccine development studies.
