Outcomes of hematopoietic stem cell gene therapy for Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, autoimmunity, and lymphoid malignancies. Gene therapy (GT) to modify autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplantation for patients who lack well-matched donors, avoiding graft-versus-host-disease. We report the outcomes of a phase 1/2 clinical trial in which 5 patients with severe WAS underwent GT using a self-inactivating lentiviral vector expressing the human WAS complementary DNA under the control of a 1.6-kB fragment of the autologous promoter after busulfan and fludarabine conditioning. All patients were alive and well with sustained multilineage vector gene marking (median follow-up: 7.6 years). Clinical improvement of eczema, infections, and bleeding diathesis was universal. Immune function was consistently improved despite subphysiologic levels of transgenic WAS protein expression. Improvements in platelet count and cytoskeletal function in myeloid cells were most prominent in patients with high vector copy number in the transduced product. Two patients with a history of autoimmunity had flares of autoimmunity after GT, despite similar percentages of WAS protein-expressing cells and gene marking to those without autoimmunity. Patients with flares of autoimmunity demonstrated poor numerical recovery of T cells and regulatory T cells (Tregs), interleukin-10-producing regulatory B cells (Bregs), and transitional B cells. Thus, recovery of the Breg compartment, along with Tregs appears to be protective against development of autoimmunity after GT. These results indicate that clinical and laboratory manifestations of WAS are improved with GT with an acceptable safety profile. This trial is registered at clinicaltrials.gov as #NCT01410825.

Lentiviral globin gene therapy with reduced-intensity conditioning in adults with ß-thalassemia: a phase 1 trial.

ß-Thalassemias are inherited anemias that are caused by the absent or insufficient production of the ß chain of hemoglobin. Here we report 6-8-year follow-up of four adult patients with transfusion-dependent ß-thalassemia who were infused with autologous CD34+ cells transduced with the TNS9.3.55 lentiviral globin vector after reduced-intensity conditioning (RIC) in a phase 1 clinical trial ( NCT01639690) . Patients were monitored for insertional mutagenesis and the generation of a replication-competent lentivirus (safety and tolerability of the infusion product after RIC-primary endpoint) and engraftment of genetically modified autologous CD34+ cells, expression of the transduced ß-globin gene and post-transplant transfusion requirements (efficacy-secondary endpoint). No unexpected safety issues occurred during conditioning and cell product infusion. Hematopoietic gene marking was very stable but low, reducing transfusion requirements in two patients, albeit not achieving transfusion independence. Our findings suggest that non-myeloablative conditioning can achieve durable stem cell engraftment but underscore a minimum CD34+ cell transduction requirement for effective therapy. Moderate clonal expansions were associated with integrations near cancer-related genes, suggestive of non-erythroid activity of globin vectors in stem/progenitor cells. These correlative findings highlight the necessity of cautiously monitoring patients harboring globin vectors.

SARS-CoV-2 variants associated with vaccine breakthrough in the Delaware Valley through summer 2021.

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed three-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of 1.25-3.18). This study thus provides a detailed picture of viral evolution in the Delaware Valley and a geographically matched analysis of vaccine breakthroughs; it also introduces a rigorous statistical approach to interrogating enrichment of viral variants.

Redondovirus Diversity and Evolution on Global, Individual, and Molecular Scales.

Redondoviridae is a newly established family of circular Rep-encoding single-stranded (CRESS) DNA viruses found in the human ororespiratory tract. Redondoviruses were previously found in ∼15% of respiratory specimens from U.S. urban subjects; levels were elevated in individuals with periodontitis or critical illness. Here, we report higher redondovirus prevalence in saliva samples: four rural African populations showed 61 to 82% prevalence, and an urban U.S. population showed 32% prevalence. Longitudinal, limiting-dilution single-genome sequencing revealed diverse strains of both redondovirus species (Brisavirus and Vientovirus) in single individuals, persistence over time, and evidence of intergenomic recombination. Computational analysis of viral genomes identified a recombination hot spot associated with a conserved potential DNA stem-loop structure. To assess the possible role of this site in recombination, we carried out in vitro studies which showed that this potential stem-loop was cleaved by the virus-encoded Rep protein. In addition, in reconstructed reactions, a Rep-DNA covalent intermediate was shown to mediate DNA strand transfer at this site. Thus, redondoviruses are highly prevalent in humans, found in individuals on multiple continents, heterogeneous even within individuals and encode a Rep protein implicated in facilitating recombination. IMPORTANCERedondoviridae is a recently established family of DNA viruses predominantly found in the human respiratory tract and associated with multiple clinical conditions. In this study, we found high redondovirus prevalence in saliva from urban North American individuals and nonindustrialized African populations in Botswana, Cameroon, Ethiopia, and Tanzania. Individuals on both continents harbored both known redondovirus species. Global prevalence of both species suggests that redondoviruses have long been associated with humans but have remained undetected until recently due to their divergent genomes. By sequencing single redondovirus genomes in longitudinally sampled humans, we found that redondoviruses persisted over time within subjects and likely evolve by recombination. The Rep protein encoded by redondoviruses catalyzes multiple reactions in vitro, consistent with a role in mediating DNA replication and recombination. In summary, we identify high redondovirus prevalence in humans across multiple continents, longitudinal heterogeneity and persistence, and potential mechanisms of redondovirus evolution by recombination.

Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons.

BACKGROUND: Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection. RESULTS: Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in "single pot" reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes. CONCLUSIONS: LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.

An integrated self-powered 3D printed sample concentrator for highly sensitive molecular detection of HIV in whole blood at the point of care.

Rapid and efficient biological sample preparation and pretreatment are crucial for highly sensitive, reliable and reproducible molecular detection of infectious diseases. Herein, we report a self-powered, integrated sample concentrator (SPISC) for rapid plasma separation, pathogen lysis, nucleic acid trapping and enrichment at the point of care. The proposed sample concentrator uses a combination of gravitational sedimentation of blood cells and capillary force for rapid, self-powered plasma separation. The pathogens (e.g., HIV virus) in separated plasma were directly lysed and pathogen nucleic acid was enriched by an integrated, flow-through FTA® membrane in the concentrator, enabling highly efficient nucleic acid preparation. The FTA® membrane of the SPISC is easy to store and transport at room temperature without need for uninterrupted cold chain, which is crucial for point of care sampling in resource-limited settings. The platform has been successfully applied to detect HIV virus in blood samples. Our experiments show that the sample concentrator can achieve a plasma separation efficiency as high as 95% and a detection sensitivity as low as 10 copies per 200 µL blood (∼100 copies per mL plasma) with variability less than 7%. The sample concentrator described is fully compatible with downstream nucleic acid detection and has great potential for early diagnostics, monitoring and management of infectious diseases at the point of care.

Long-term outcomes after gene therapy for adenosine deaminase severe combined immune deficiency.

Patients lacking functional adenosine deaminase activity have severe combined immunodeficiency (ADA SCID), which can be treated with ADA enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT), or autologous HSCT with gene-corrected cells (gene therapy [GT]). A cohort of 10 ADA SCID patients, aged 3 months to 15 years, underwent GT in a phase 2 clinical trial between 2009 and 2012. Autologous bone marrow CD34+ cells were transduced ex vivo with the MND (myeloproliferative sarcoma virus, negative control region deleted, dl587rev primer binding site)-ADA gammaretroviral vector (gRV) and infused following busulfan reduced-intensity conditioning. These patients were monitored in a long-term follow-up protocol over 8 to 11 years. Nine of 10 patients have sufficient immune reconstitution to protect against serious infections and have not needed to resume ERT or proceed to secondary allogeneic HSCT. ERT was restarted 6 months after GT in the oldest patient who had no evidence of benefit from GT. Four of 9 evaluable patients with the highest gene marking and B-cell numbers remain off immunoglobulin replacement therapy and responded to vaccines. There were broad ranges of responses in normalization of ADA enzyme activity and adenine metabolites in blood cells and levels of cellular and humoral immune reconstitution. Outcomes were generally better in younger patients and those receiving higher doses of gene-marked CD34+ cells. No patient experienced a leukoproliferative event after GT, despite persisting prominent clones with vector integrations adjacent to proto-oncogenes. These long-term findings demonstrate enduring efficacy of GT for ADA SCID but also highlight risks of genotoxicity with gRVs. This trial was registered at www.clinicaltrials.gov as #NCT00794508.

SARS-CoV-2 Genomic Variation in Space and Time in Hospitalized Patients in Philadelphia.

The severe acute respiratory coronavirus 2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. The epidemic accelerated in Philadelphia, PA, in the spring of 2020, with the city experiencing a first peak of infections on 15 April, followed by a decline through midsummer. Here, we investigate spread of the epidemic in the first wave in Philadelphia using full-genome sequencing of 52 SARS-CoV-2 samples obtained from 27 hospitalized patients collected between 30 March and 17 July 2020. Sequences most commonly resembled lineages circulating at earlier times in New York, suggesting transmission primarily from this location, though a minority of Philadelphia genomes matched sequences from other sites, suggesting additional introductions. Multiple genomes showed even closer matches to other Philadelphia isolates, suggestive of ongoing transmission within Philadelphia. We found that all of our isolates contained the D614G substitution in the viral spike and belong to lineages variously designated B.1, Nextstrain clade 20A or 20C, and GISAID clade G or GH. There were no viral sequence polymorphisms detectably associated with disease outcome. For some patients, genome sequences were determined longitudinally or concurrently from multiple body sites. In both cases, some comparisons showed reproducible polymorphisms, suggesting initial seeding with multiple variants and/or accumulation of polymorphisms after infection. These results thus provide data on the sources of SARS-CoV-2 infection in Philadelphia and begin to explore the dynamics within hospitalized patients.IMPORTANCE Understanding how SARS-CoV-2 spreads globally and within infected individuals is critical to the development of mitigation strategies. We found that most lineages in Philadelphia had resembled sequences from New York, suggesting infection primarily but not exclusively from this location. Many genomes had even nearer neighbors within Philadelphia, indicating local spread. Multiple genome sequences were available for some subjects and in a subset of cases could be shown to differ between time points and body sites within an individual, indicating heterogeneous viral populations within individuals and raising questions on the mechanisms responsible. There was no evidence that different lineages were associated with different outcomes in patients, emphasizing the importance of individual-specific vulnerability.

SARS-CoV-2 Variants Associated with Vaccine Breakthrough in the Delaware Valley through Summer 2021.

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here, we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed 3-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of1.25-3.18). This study thus overviews viral evolution and vaccine breakthroughs in the Delaware Valley and introduces a rigorous statistical approach to interrogating enrichment of breakthrough variants against a changing background. IMPORTANCE SARS-CoV-2 vaccination is highly effective at reducing viral infection, hospitalization and death. However, vaccine breakthrough infections have been widely observed, raising the question of whether particular viral variants or viral mutations are associated with breakthrough. Here, we report analysis of 2621 surveillance isolates from people diagnosed with COVID-19 in the Delaware Valley in southeastern Pennsylvania, allowing rigorous comparison to 159 vaccine breakthrough case specimens. Our best estimate is a 3-fold enrichment for some lineages of delta among breakthroughs, and enrichment of a notable spike substitution, N501Y. We introduce statistical methods that should be widely useful for evaluating vaccine breakthroughs and other viral phenotypes.

A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells.

Nine dogs with hemophilia A were treated with adeno-associated viral (AAV) gene therapy and followed for up to 10 years. Administration of AAV8 or AAV9 vectors expressing canine factor VIII (AAV-cFVIII) corrected the FVIII deficiency to 1.9-11.3% of normal FVIII levels. In two of nine dogs, levels of FVIII activity increased gradually starting about 4 years after treatment. None of the dogs showed evidence of tumors or altered liver function. Analysis of integration sites in liver samples from six treated dogs identified 1,741 unique AAV integration events in genomic DNA and expanded cell clones in five dogs, with 44% of the integrations near genes involved in cell growth. All recovered integrated vectors were partially deleted and/or rearranged. Our data suggest that the increase in FVIII protein expression in two dogs may have been due to clonal expansion of cells harboring integrated vectors. These results support the clinical development of liver-directed AAV gene therapy for hemophilia A, while emphasizing the importance of long-term monitoring for potential genotoxicity.

Lack of detection of a human placenta microbiome in samples from preterm and term deliveries.

BACKGROUND: Historically, the human womb has been thought to be sterile in healthy pregnancies, but this idea has been challenged by recent studies using DNA sequence-based methods, which have suggested that the womb is colonized with bacteria. For example, analysis of DNA from placenta samples yielded small proportions of microbial sequences which were proposed to represent normal bacterial colonization. However, an analysis by our group showed no distinction between background negative controls and placenta samples. Also supporting the idea that the womb is sterile is the observation that germ-free mammals can be generated by sterile delivery of neonates into a sterile isolator, after which neonates remain germ-free, which would seem to provide strong data in support of sterility of the womb. RESULTS: To probe this further and to investigate possible placental colonization associated with spontaneous preterm birth, we carried out another study comparing microbiota in placenta samples from 20 term and 20 spontaneous preterm deliveries. Both 16S rRNA marker gene sequencing and shotgun metagenomic sequencing were used to characterize placenta and control samples. We first quantified absolute amounts of bacterial 16S rRNA gene sequences using 16S rRNA gene quantitative PCR (qPCR). As in our previous study, levels were found to be low in the placenta samples and indistinguishable from negative controls. Analysis by DNA sequencing did not yield a placenta microbiome distinct from negative controls, either using marker gene sequencing as in our previous work, or with shotgun metagenomic sequencing. Several types of artifacts, including erroneous read classifications and barcode misattribution, needed to be identified and removed from the data to clarify this point. CONCLUSIONS: Our findings do not support the existence of a consistent placental microbiome, in either placenta from term deliveries or spontaneous preterm births.

Single Cell Bottlenecks in the Pathogenesis of Streptococcus pneumoniae.

Herein, we studied a virulent isolate of the leading bacterial pathogen Streptococcus pneumoniae in an infant mouse model of colonization, disease and transmission, both with and without influenza A (IAV) co-infection. To identify vulnerable points in the multiple steps involved in pneumococcal pathogenesis, this model was utilized for a comprehensive analysis of population bottlenecks. Our findings reveal that in the setting of IAV co-infection the organism must pass through single cell bottlenecks during bloodstream invasion from the nasopharynx within the host and in transmission between hosts. Passage through these bottlenecks was not associated with genetic adaptation by the pathogen. The bottleneck in transmission occurred between bacterial exit from one host and establishment in another explaining why the number of shed organisms in secretions is critical to overcoming it. These observations demonstrate how viral infection, and TLR-dependent innate immune responses it stimulates and that are required to control it, drive bacterial contagion.

Comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota.

BACKGROUND: Recent studies have suggested that bacteria associated with the placenta-a "placental microbiome"-may be important in reproductive health and disease. However, a challenge in working with specimens with low bacterial biomass, such as placental samples, is that some or all of the bacterial DNA may derive from contamination in dust or commercial reagents. To investigate this, we compared placental samples from healthy deliveries to a matched set of contamination controls, as well as to oral and vaginal samples from the same women. RESULTS: We quantified total 16S rRNA gene copies using quantitative PCR and found that placental samples and negative controls contained low and indistinguishable copy numbers. Oral and vaginal swab samples, in contrast, showed higher copy numbers. We carried out 16S rRNA gene sequencing and community analysis and found no separation between communities from placental samples and contamination controls, though oral and vaginal samples showed characteristic, distinctive composition. Two different DNA purification methods were compared with similar conclusions, though the composition of the contamination background differed. Authentically present microbiota should yield mostly similar results regardless of the purification method used-this was seen for oral samples, but no placental bacterial lineages were (1) shared between extraction methods, (2) present at >1 % of the total, and (3) present at greater abundance in placental samples than contamination controls. CONCLUSIONS: We conclude that for this sample set, using the methods described, we could not distinguish between placental samples and contamination introduced during DNA purification.

Peptidoglycan from the gut microbiota governs the lifespan of circulating phagocytes at homeostasis.

Maintenance of myeloid cell homeostasis requires continuous turnover of phagocytes from the bloodstream, yet whether environmental signals influence phagocyte longevity in the absence of inflammation remains unknown. Here, we show that the gut microbiota regulates the steady-state cellular lifespan of neutrophils and inflammatory monocytes, the 2 most abundant circulating myeloid cells and key contributors to inflammatory responses. Treatment of mice with broad-spectrum antibiotics, or with the gut-restricted aminoglycoside neomycin alone, accelerated phagocyte turnover and increased the rates of their spontaneous apoptosis. Metagenomic analyses revealed that neomycin altered the abundance of intestinal bacteria bearing γ-d-glutamyl-meso-diaminopimelic acid, a ligand for the intracellular peptidoglycan sensor Nod1. Accordingly, signaling through Nod1 was both necessary and sufficient to mediate the stimulatory influence of the flora on myeloid cell longevity. Stimulation of Nod1 signaling increased the frequency of lymphocytes in the murine intestine producing the proinflammatory cytokine interleukin 17A (IL-17A), and liberation of IL-17A was required for transmission of Nod1-dependent signals to circulating phagocytes. Together, these results define a mechanism through which intestinal microbes govern a central component of myeloid homeostasis and suggest perturbations of commensal communities can influence steady-state regulation of cell fate.

Bacterial exploitation of phosphorylcholine mimicry suppresses inflammation to promote airway infection.

Regulation of neutrophil activity is critical for immune evasion among extracellular pathogens, yet the mechanisms by which many bacteria disrupt phagocyte function remain unclear. Here, we have shown that the respiratory pathogen Streptococcus pneumoniae disables neutrophils by exploiting molecular mimicry to degrade platelet-activating factor (PAF), a host-derived inflammatory phospholipid. Using mass spectrometry and murine upper airway infection models, we demonstrated that phosphorylcholine (ChoP) moieties that are shared by PAF and the bacterial cell wall allow S. pneumoniae to leverage a ChoP-remodeling enzyme (Pce) to remove PAF from the airway. S. pneumoniae-mediated PAF deprivation impaired viability, activation, and bactericidal capacity among responding neutrophils. In the absence of Pce, neutrophils rapidly cleared S. pneumoniae from the airway and impeded invasive disease and transmission between mice. Abrogation of PAF signaling rendered Pce dispensable for S. pneumoniae persistence, reinforcing that this enzyme deprives neutrophils of essential PAF-mediated stimulation. Accordingly, exogenous activation of neutrophils overwhelmed Pce-mediated phagocyte disruption. Haemophilus influenzae also uses an enzyme, GlpQ, to hydrolyze ChoP and subvert PAF function, suggesting that mimicry-driven immune evasion is a common paradigm among respiratory pathogens. These results identify a mechanism by which shared molecular structures enable microbial enzymes to subvert host lipid signaling, suppress inflammation, and ensure bacterial persistence at the mucosa.

Macrophage Migration Inhibitory Factor Is Detrimental in Pneumococcal Pneumonia and a Target for Therapeutic Immunomodulation.

Mortality from pneumococcal pneumonia remains high despite antibiotic therapy, highlighting the pathogenic potential for host inflammation. We demonstrate that macrophage migration inhibitory factor (MIF), an innate immune mediator, is detrimental for survival and associated with lung pathology, inflammatory cellular infiltration, and bacterial replication in a mouse model of pneumococcal pneumonia, despite being necessary for clearance from the nasopharynx. Treatment of animals with a small-molecule inhibitor of MIF improves survival by reducing inflammation and improving bacterial control. Our work demonstrates that MIF modulates beneficial versus detrimental inflammatory responses in the host-pneumococcal interaction and is a potential target for therapeutic modulation.

Influenza promotes pneumococcal growth during coinfection by providing host sialylated substrates as a nutrient source.

Much of the mortality attributed to influenza virus is due to secondary bacterial pneumonia, particularly from Streptococcus pneumoniae. However, mechanisms underlying this coinfection are incompletely understood. We find that prior influenza infection enhances pneumococcal colonization of the murine nasopharynx, which in turn promotes bacterial spread to the lungs. Influenza accelerates bacterial replication in vivo, and sialic acid, a major component of airway glycoconjugates, is identified as the host-derived metabolite that stimulates pneumococcal proliferation. Influenza infection increases sialic acid and sialylated mucin availability and enhances desialylation of host glycoconjugates. Pneumococcal genes for sialic acid catabolism are required for influenza to promote bacterial growth. Decreasing sialic acid availability in vivo by genetic deletion of the major airway mucin Muc5ac or mucolytic treatment limits influenza-induced pneumococcal replication. Our findings suggest that higher rates of disease during coinfection could stem from influenza-provided sialic acid, which increases pneumococcal proliferation, colonization, and aspiration.

MARCO is required for TLR2- and Nod2-mediated responses to Streptococcus pneumoniae and clearance of pneumococcal colonization in the murine nasopharynx.

Streptococcus pneumoniae is a common human pathogen that accounts for >1 million deaths every year. Colonization of the nasopharynx by S. pneumoniae precedes pulmonary and other invasive diseases and, therefore, is a promising target for intervention. Because the receptors scavenger receptor A (SRA), macrophage receptor with collagenous structure (MARCO), and mannose receptor (MR) have been identified as nonopsonic receptors for S. pneumoniae in the lung, we used scavenger receptor knockout mice to study the roles of these receptors in the clearance of S. pneumoniae from the nasopharynx. MARCO(-/-), but not SRA(-/-) or MR(-/-), mice had significantly impaired clearance of S. pneumoniae from the nasopharynx. In addition to impairment in bacterial clearance, MARCO(-/-) mice had abrogated cytokine production and cellular recruitment to the nasopharynx following colonization. Furthermore, macrophages from MARCO(-/-) mice were deficient in cytokine and chemokine production, including type I IFNs, in response to S. pneumoniae. MARCO was required for maximal TLR2- and nucleotide-binding oligomerization domain-containing (Nod)2-dependent NF-κB activation and signaling that ultimately resulted in clearance. Thus, MARCO is an important component of anti-S. pneumoniae responses in the murine nasopharynx during colonization.

Pneumococcal surface protein A inhibits complement deposition on the pneumococcal surface by competing with the binding of C-reactive protein to cell-surface phosphocholine.

In the presence of normal serum, complement component C3 is deposited on pneumococci primarily via the classical pathway. Pneumococcal surface protein A (PspA), a major virulence factor of pneumococci, effectively inhibits C3 deposition. PspA's C terminus has a choline-binding domain that anchors PspA to the phosphocholine (PC) moieties on the pneumococcal surface. C-reactive protein (CRP), another important host defense molecule, also binds to PC, and CRP binding to pneumococci enhances complement C3 deposition through the classical pathway. Using flow cytometry of PspA(+) and PspA(-) strains, we observed that the absence of PspA led to exposure of PC, enhanced the surface binding of CRP, and increased the deposition of C3. Moreover, when the PspA(-) mutant was incubated with a pneumococcal eluate containing native PspA, there was decreased deposition of CRP and C3 on the pneumococcal surface compared with incubation with an eluate from a PspA(-) strain. This inhibition was not observed when a recombinant PspA fragment, which lacks the choline-binding region of PspA, was added to the PspA(-) mutant. Also, there was much greater C3 deposition onto the PspA(-) pneumococcus when exposed to normal mouse serum from wild-type mice as compared with that from CRP knockout mice. Furthermore, when CRP knockout mouse serum was replenished with CRP, there was a dose-dependent increase in C3 deposition. The combined data reveal a novel mechanism of complement inhibition by a bacterial protein: inhibition of CRP surface binding and, thus, diminution of CRP-mediated complement deposition.

Identification of the targets of cross-reactive antibodies induced by Streptococcus pneumoniae colonization.

Much of the efficacy of current pneumococcal conjugate vaccines lies in their ability to decrease carriage of vaccine serotypes in the population. Novel and more-broadly acting vaccines would also need to target carriage in order to be as effective. We have previously shown that model murine carriage of Streptococcus pneumoniae can elicit antibody-dependent immunity and can protect against a virulent heterologous challenge strain. This study set out to identify S. pneumoniae surface antigens that may elicit cross-reactive antibodies following colonization. Western blot analysis using sera from colonized mice identified the previously characterized immunogens pneumococcal surface protein A (PspA), putative proteinase maturation protein A (PpmA), and pneumococcal surface adhesin A (PsaA) as such antigens. Using flow cytometry, PspA was found to be the major target of surface-bound cross-reactive IgG in sera from TIGR4 Delta cps-colonized mice, with a modest contribution from PpmA and none from PsaA. In human sera, however, only mutants lacking PpmA were shown to have reduced binding of surface IgG compared to wild-type strains, suggesting that prior exposure to S. pneumoniae in humans may induce PpmA antibodies. We also investigated if cross-reactive antibodies induced by these antigens may be cross-protective against carriage. Despite the immunogenicity of PspA, PpmA, and PsaA, mice were still protected following colonization with mutants lacking these antigens, suggesting they are not necessary for cross-protection induced by carriage. Our findings suggest that a whole-organism approach may be needed to broadly diminish carriage.