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RationaleNasopharyngeal 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.ObjectivesTo assess protective efficacy of nasopharyngeal administration of unencapsulated S. pneumoniae strains in murine infection models.MethodsStrains 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 resultsThe ∆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.ConclusionsColonisation 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.

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 pneumoniae. B cell depletion of naïve C57Bl/6 mice reduced natural IgM recognition of S. pneumoniae, but did not increase susceptibility to S. pneumoniae pneumonia. ELISA and flow cytometry assays demonstrated significantly reduced IgG and IgM recognition of S. pneumoniae in sera from mice treated with B cell depletion prior to S. pneumoniae nasopharyngeal colonization compared to untreated mice. Colonization induced antibody responses to protein rather than capsular antigen, and when measured using a protein array B cell depletion prior to colonization reduced serum levels of IgG to several protein antigens. However, B cell depleted S. pneumoniae colonized mice were still partially protected against both lung infection and septicemia when challenged with S. pneumoniae after reconstitution of their B cells. These data indicate that although B cell depletion markedly impairs antibody recognition of S. pneumoniae in colonized mice, some protective immunity is maintained, perhaps mediated by cellular immunity.

Objectives: Patients with rheumatoid arthritis (RA) have an increased susceptibility to infections, 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 characterised in serum from RA patients and disease controls (myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)) using whole‐cell ELISA, a flow cytometry opsonisation assay and an S. pneumoniae protein array. For the RA patients, results were compared before and after B‐cell depletion therapy. Results: Compared to a well‐characterised 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 opsonisation 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

An ex vivo, porcine spleen perfusion model was established to study the early events occurring in the spleen prior to the onset of bacterial sepsis, using organs retrieved from animals slaughtered for food production. Porcine spleens were harvested from adult pigs and connected to a normothermic extracorporeal perfusion circuit. Constant perfusion of heparinized blood was performed for 6 hours. After injection of Streptococcus pneumoniae to the circuit, serial samples of both blood and spleen biopsies were collected and analyzed. Functionality of the perfused organs was assessed by monitoring the blood-gas parameters, flow rate and filtering capability of the organ. Interestingly, we observed full clearance of bacteria from the blood and an increase in bacterial counts in the spleen. Classical histology and immunohistochemistry on biopsies also confirmed no major damage in the organ architecture and no changes in the immune cell distribution other than the presence of clusters of pneumococci. A time-course study confirmed that each focus of infection derived from the replication of single pneumococcal cells within splenic macrophages. The model proposed - in line with the 3Rs principles - has utility in the replacement of experimental animals in infection research. Murine models are prevalently used to study pneumococcal infections but are often not predictive for humans due to substantial differences in the immune systems of the two species. This model is designed to overcome these limitations, since porcine immunology, and splenic architecture in particular, closely resemble those of humans. [ABSTRACT FROM AUTHOR]

Virulence screens have indicated potential roles during Streptococcus pneumoniae infection for the one-carbon metabolism pathway component Fhs and proline synthesis mediated by ProABC. To define how these metabolic pathways affect S. pneumoniae virulence, we have investigated the phenotypes, transcription, and metabolic profiles of Δ fhs and Δ proABC mutants. S. pneumoniae capsular serotype 6B BHN418 Δ fhs and Δ proABC mutant strains had strongly reduced virulence in mouse sepsis and pneumonia models but could colonize the nasopharynx. Both mutant strains grew normally in complete media but had markedly impaired growth in chemically defined medium, human serum, and human cerebrospinal fluid. The BHN418 Δ proABC strain also had impaired growth under conditions of osmotic and oxidative stress. The virulence role of proABC was strain specific, as the D39 Δ proABC strain could still cause septicemia and grow in serum. Compared to culture in broth, in serum, the BHN418 Δ fhs and Δ proABC strains showed considerable derangement in global gene transcription that affected multiple but different metabolic pathways for each mutant strain. Metabolic data suggested that Δ fhs had an impaired stringent response, and when cultured in sera, BHN418 Δ fhs and Δ proABC were under increased oxidative stress and had altered lipid profiles. Loss of proABC also affected carbohydrate metabolism and the accumulation of peptidoglycan synthesis precursors in the BHN418 but not the D39 background, linking this phenotype to the conditional virulence phenotype. These data identify the S. pneumoniae metabolic functions affected by S. pneumoniae one-carbon metabolism and proline biosynthesis, and the role of these genetic loci for establishing systemic infection.IMPORTANCERapid adaptation to grow within the physiological conditions found in the host environment is an essential but poorly understood virulence requirement for systemic pathogens such as Streptococcus pneumoniae . We have now demonstrated an essential role for the one-carbon metabolism pathway and a conditional role depending on strain background for proline biosynthesis for S. pneumoniae growth in serum or cerebrospinal fluid, and therefore for systemic virulence. RNAseq and metabolomic data demonstrated that the loss of one-carbon metabolism or proline biosynthesis has profound but differing effects on S. pneumoniae metabolism in human serum, identifying the metabolic processes dependent on each pathway during systemic infection. These data provide a more detailed understanding of the adaptations required by systemic bacterial pathogens in order to cause infection and demonstrate that the requirement for some of these adaptations varies between strains from the same species and could therefore underpin strain variations in virulence potential.

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., Competing Interests: GW was employed by the company Novartis Institute for BioMedical Research. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)