Identification of mouse CD4+ T cell epitopes in SARS-CoV-2 BA.1 spike and nucleocapsid for use in peptide:MHCII tetramers.

Understanding adaptive immunity against SARS-CoV-2 is a major requisite for the development of effective vaccines and treatments for COVID-19. CD4+ T cells play an integral role in this process primarily by generating antiviral cytokines and providing help to antibody-producing B cells. To empower detailed studies of SARS-CoV-2-specific CD4+ T cell responses in mouse models, we comprehensively mapped I-Ab-restricted epitopes for the spike and nucleocapsid proteins of the BA.1 variant of concern via IFNγ ELISpot assay. This was followed by the generation of corresponding peptide:MHCII tetramer reagents to directly stain epitope-specific T cells. Using this rigorous validation strategy, we identified 6 immunogenic epitopes in spike and 3 in nucleocapsid, all of which are conserved in the ancestral Wuhan strain. We also validated a previously identified epitope from Wuhan that is absent in BA.1. These epitopes and tetramers will be invaluable tools for SARS-CoV-2 antigen-specific CD4+ T cell studies in mice.

Structure-Based Discovery of Lipoteichoic Acid Synthase Inhibitors.

Lipoteichoic acid synthase (LtaS) is a key enzyme for the cell wall biosynthesis of Gram-positive bacteria. Gram-positive bacteria that lack lipoteichoic acid (LTA) exhibit impaired cell division and growth defects. Thus, LtaS appears to be an attractive antimicrobial target. The pharmacology around LtaS remains largely unexplored with only two small-molecule LtaS inhibitors reported, namely "compound 1771" and the Congo red dye. Structure-based drug discovery efforts against LtaS remain unattempted due to the lack of an inhibitor-bound structure of LtaS. To address this, we combined the use of a molecular docking technique with molecular dynamics (MD) simulations to model a plausible binding mode of compound 1771 to the extracellular catalytic domain of LtaS (eLtaS). The model was validated using alanine mutagenesis studies combined with isothermal titration calorimetry. Additionally, lead optimization driven by our computational model resulted in an improved version of compound 1771, namely, compound 4 which showed greater affinity for binding to eLtaS than compound 1771 in biophysical assays. Compound 4 reduced LTA production in S. aureus dose-dependently, induced aberrant morphology as seen for LTA-deficient bacteria, and significantly reduced bacteria titers in the lung of mice infected with S. aureus. Analysis of our MD simulation trajectories revealed the possible formation of a transient cryptic pocket in eLtaS. Virtual screening (VS) against the cryptic pocket led to the identification of a new class of inhibitors that could potentiate ß-lactams against methicillin-resistant S. aureus. Our overall workflow and data should encourage further drug design campaign against LtaS. Finally, our work reinforces the importance of considering protein conformational flexibility to a successful VS endeavor.

Lower Density and Shorter Duration of Nasopharyngeal Carriage by Pneumococcal Serotype 1 (ST217) May Explain Its Increased Invasiveness over Other Serotypes.

Streptococcus pneumoniae is a frequent colonizer of the human nasopharynx and a major cause of life-threating invasive infections such as pneumonia, meningitis and sepsis. Over 1 million people die every year due to invasive pneumococcal disease (IPD), mainly in developing countries. Serotype 1 is a common cause of IPD; however, unlike other serotypes, it is rarely found in the carrier state in the nasopharynx, which is often considered a prerequisite for disease. The aim of this study was to understand this dichotomy. We used murine models of carriage and IPD to characterize the pathogenesis of African serotype 1 (sequence type 217) pneumococcal strains obtained from the Queen Elizabeth Central Hospital in Blantyre, Malawi. We found that ST217 pneumococcal strains were highly virulent in a mouse model of invasive pneumonia, but in contrast to the generally accepted assumption, can also successfully establish nasopharyngeal carriage. Interestingly, we found that cocolonizing serotypes may proliferate in the presence of serotype 1, suggesting that acquisition of serotype 1 carriage could increase the risk of developing IPD by other serotypes. RNA sequencing analysis confirmed that key virulence genes associated with inflammation and tissue invasiveness were upregulated in serotype 1. These data reveal important new insights into serotype 1 pathogenesis, with implications for carriage potential and risk of invasive disease through interactions with other cocolonizing serotypes, an often-overlooked factor in transmission and disease progression.IMPORTANCE The pneumococcus causes serious diseases such as pneumonia, sepsis, and meningitis and is a major cause of morbidity and mortality worldwide. Serotype 1 accounts for the majority of invasive pneumococcal disease cases in sub-Saharan Africa but is rarely found during nasopharyngeal carriage. Understanding the mechanisms leading to nasopharyngeal carriage and invasive disease by this serotype can help reduce its burden on health care systems worldwide. In this study, we also uncovered the potential impact of serotype 1 on disease progression of other coinfecting serotypes, which can have important implications for vaccine efficacy. Understanding the interactions between different serotypes during nasopharyngeal carriage may lead to improved intervention methods and therapies to reduce pneumococcal invasive disease levels.

Astrocyte- and Neuron-Derived CXCL1 Drives Neutrophil Transmigration and Blood-Brain Barrier Permeability in Viral Encephalitis.

Herpes simplex virus (HSV)-1 encephalitis has significant morbidity partly because of an over-exuberant immune response characterized by leukocyte infiltration into the brain and increased blood-brain barrier (BBB) permeability. Determining the role of specific leukocyte subsets and the factors that mediate their recruitment into the brain is critical to developing targeted immune therapies. In a murine model, we find that the chemokines CXCL1 and CCL2 are induced in the brain following HSV-1 infection. Ccr2 (CCL2 receptor)-deficient mice have reduced monocyte recruitment, uncontrolled viral replication, and increased morbidity. Contrastingly, Cxcr2 (CXCL1 receptor)-deficient mice exhibit markedly reduced neutrophil recruitment, BBB permeability, and morbidity, without influencing viral load. CXCL1 is produced by astrocytes in response to HSV-1 and by astrocytes and neurons in response to IL-1α, and it is the critical ligand required for neutrophil transendothelial migration, which correlates with BBB breakdown. Thus, the CXCL1-CXCR2 axis represents an attractive therapeutic target to limit neutrophil-mediated morbidity in HSV-1 encephalitis.

Increased pathogenicity of pneumococcal serotype 1 is driven by rapid autolysis and release of pneumolysin.

Streptococcus pneumoniae serotype 1 is the predominant cause of invasive pneumococcal disease in sub-Saharan Africa, but the mechanism behind its increased invasiveness is not well understood. Here, we use mouse models of lung infection to identify virulence factors associated with severe bacteraemic pneumonia during serotype-1 (ST217) infection. We use BALB/c mice, which are highly resistant to pneumococcal pneumonia when infected with other serotypes. However, we observe 100% mortality and high levels of bacteraemia within 24 hours when BALB/c mice are intranasally infected with ST217. Serotype 1 produces large quantities of pneumolysin, which is rapidly released due to high levels of bacterial autolysis. This leads to substantial levels of cellular cytotoxicity and breakdown of tight junctions between cells, allowing a route for rapid bacterial dissemination from the respiratory tract into the blood. Thus, our results offer an explanation for the increased invasiveness of serotype 1.

Impaired Alanine Transport or Exposure to d-Cycloserine Increases the Susceptibility of MRSA to ß-lactam Antibiotics.

Prolonging the clinical effectiveness of ß-lactams, which remain first-line antibiotics for many infections, is an important part of efforts to address antimicrobial resistance. We report here that inactivation of the predicted d-cycloserine (DCS) transporter gene cycA resensitized methicillin-resistant Staphylococcus aureus (MRSA) to ß-lactam antibiotics. The cycA mutation also resulted in hypersusceptibility to DCS, an alanine analogue antibiotic that inhibits alanine racemase and d-alanine ligase required for d-alanine incorporation into cell wall peptidoglycan. Alanine transport was impaired in the cycA mutant, and this correlated with increased susceptibility to oxacillin and DCS. The cycA mutation or exposure to DCS were both associated with the accumulation of muropeptides with tripeptide stems lacking the terminal d-ala-d-ala and reduced peptidoglycan cross-linking, prompting us to investigate synergism between ß-lactams and DCS. DCS resensitized MRSA to ß-lactams in vitro and significantly enhanced MRSA eradication by oxacillin in a mouse bacteremia model. These findings reveal alanine transport as a new therapeutic target to enhance the susceptibility of MRSA to ß-lactam antibiotics.

Dectin-2-induced CCL2 production in tissue-resident macrophages ignites cardiac arteritis.

Environmental triggers, including those from pathogens, are thought to play an important role in triggering autoimmune diseases, such as vasculitis, in genetically susceptible individuals. The mechanism by which activation of the innate immune system contributes to vessel-specific autoimmunity in vasculitis is not known. Systemic administration of Candida albicans water-soluble extract (CAWS) induces vasculitis in the aortic root and coronary arteries of mice that mimics human Kawasaki disease. We found that Dectin-2 signaling in macrophages resident in the aortic root of the heart induced early CCL2 production and the initial recruitment of CCR2+ inflammatory monocytes (iMo) into the aortic root and coronary arteries. iMo differentiated into monocyte-derived dendritic cells (Mo-DC) in the vessel wall and were induced to release IL-1ß in a Dectin-2-Syk-NLRP3 inflammasome dependent pathway. IL-1ß then activated cardiac endothelial cells to express CXCL1 and CCL2 and adhesion molecules that induced neutrophil and further iMo recruitment and accumulation in the aortic root and coronary arteries. Our findings demonstrate that Dectin-2-mediated induction of CCL2 production by macrophages resident in the aortic root and coronary arteries initiates vascular inflammation in a model of Kawasaki disease, suggesting an important role for the innate immune system in initiating vasculitis.

Evolutionary trade-offs associated with loss of PmrB function in host-adapted Pseudomonas aeruginosa.

Pseudomonas aeruginosa colonises the upper airway of cystic fibrosis (CF) patients, providing a reservoir of host-adapted genotypes that subsequently establish chronic lung infection. We previously experimentally-evolved P. aeruginosa in a murine model of respiratory tract infection and observed early-acquired mutations in pmrB, encoding the sensor kinase of a two-component system that promoted establishment and persistence of infection. Here, using proteomics, we show downregulation of proteins involved in LPS biosynthesis, antimicrobial resistance and phenazine production in pmrB mutants, and upregulation of proteins involved in adherence, lysozyme resistance and inhibition of the chloride ion channel CFTR, relative to wild-type strain LESB65. Accordingly, pmrB mutants are susceptible to antibiotic treatment but show enhanced adherence to airway epithelial cells, resistance to lysozyme treatment, and downregulate host CFTR expression. We propose that P. aeruginosa pmrB mutations in CF patients are subject to an evolutionary trade-off, leading to enhanced colonisation potential, CFTR inhibition, and resistance to host defences, but also to increased susceptibility to antibiotics.

Comparative Genomic Analysis and In Vivo Modeling of Streptococcus pneumoniae ST3081 and ST618 Isolates Reveal Key Genetic and Phenotypic Differences Contributing to Clonal Replacement of Serotype 1 in The Gambia.

Streptococcus pneumoniae serotype 1 is one of the leading causes of invasive pneumococcal disease (IPD) in West Africa, with ST618 being the dominant cause of IPD in The Gambia. Recently however, a rare example of clonal replacement was observed, where the ST3081 clone of serotype 1 replaced the predominant ST618 clone as the main cause of IPD. In the current study, we sought to find the reasons for this unusual replacement event. Using whole-genome sequence analysis and clinically relevant models of in vivo infection, we identified distinct genetic and phenotypic characteristics of the emerging ST3081 clone. We show that ST3081 is significantly more virulent than ST618 in models of invasive pneumonia, and is carried at higher densities than ST618 during nasopharyngeal carriage. We also observe sequence type-specific accessory genes and a unique sequence type-specific fixed mutation in the pneumococcal toxin pneumolysin, which is associated with increased hemolytic activity in ST3081 and may contribute to increased virulence in this clone. Our study provides evidence that, within the same serotype 1 clonal complex, biological properties differ significantly from one clone to another in terms of virulence and host invasiveness, and that these differences may be the result of key genetic differences within the genome.

Epidemiological and Molecular Characterization of an Invasive Group A Streptococcus emm32.2 Outbreak.

An emm32.2 invasive group A streptococcus (iGAS) outbreak occurred in Liverpool from January 2010 to September 2012. This genotype had not previously been identified in Liverpool, but was responsible for 32% (14/44) of all iGAS cases reported during this time period. We performed a case-case comparison of emm32.2 iGAS cases with non-emm32.2 control iGAS cases identified in the Liverpool population over the same time period to assess patient risk factors for emm32.2 iGAS infection. The emm32.2 iGAS cases were confined to the adult population. We show that homelessness, intravenous drug use, and alcohol abuse predisposed patients to emm32.2 iGAS disease; however, no obvious epidemiological linkage between the patients with emm32.2 iGAS could be identified. Comparative whole-genome sequencing analysis of emm32.2 iGAS and non-emm32.2 control isolates was also performed to identify pathogen factors which might have driven the outbreak. We identified 19 genes, five of which had previously been implicated in virulence, which were present in all of the emm32.2 iGAS isolates but not present in any of the non-emm32.2 control isolates. We report that a novel emm32.2 genotype emerged in Liverpool in 2010 and identified a specific subset of genes, which could have allowed this novel emm32.2 genotype to persist in a disadvantaged population in the region over a 3-year period.

Airborne dust and high temperatures are risk factors for invasive bacterial disease.

BACKGROUND: The Sahel region of West Africa has the highest bacterial meningitis attack and case fatality rate in the world. The effect of climatic factors on patterns of invasive respiratory bacterial disease is not well documented. OBJECTIVE: We aimed to assess the link between climatic factors and occurrence of invasive respiratory bacterial disease in a Sahel region of Niger. METHODS: We conducted daily disease surveillance and climatic monitoring over an 8-year period between January 1, 2003, and December 31, 2010, in Niamey, Niger, to determine risk factors for bacterial meningitis and invasive bacterial disease. We investigated the mechanistic effects of these factors on Streptococcus pneumoniae infection in mice. RESULTS: High temperatures and low visibility (resulting from high concentrations of airborne dust) were identified as significant risk factors for bacterial meningitis. Dust inhalation or exposure to high temperatures promoted progression of stable asymptomatic pneumococcal nasopharyngeal carriage to pneumonia and invasive disease. Dust exposure significantly reduced phagocyte-mediated bacterial killing, and exposure to high temperatures increased release of the key pneumococcal toxin pneumolysin through increased bacterial autolysis. CONCLUSION: Our findings show that climatic factors can have a substantial influence on infectious disease patterns, altering density of pneumococcal nasopharyngeal carriage, reducing phagocytic killing, and resulting in increased inflammation and tissue damage and consequent invasiveness. Climatic surveillance should be used to forecast invasive bacterial disease epidemics, and simple control measures to reduce particulate inhalation might reduce the incidence of invasive bacterial disease in regions of the world exposed to high temperatures and increased airborne dust.

Understanding pneumococcal serotype 1 biology through population genomic analysis.

BACKGROUND: Pneumococcus kills over one million children annually and over 90 % of these deaths occur in low-income countries especially in Sub-Saharan Africa (SSA) where HIV exacerbates the disease burden. In SSA, serotype 1 pneumococci particularly the endemic ST217 clone, causes majority of the pneumococcal disease burden. To understand the evolution of the virulent ST217 clone, we analysed ST217 whole genomes from isolates sampled from African and Asian countries. METHODS: We analysed 226 whole genome sequences from the ST217 lineage sampled from 9 African and 4 Asian countries. We constructed a whole genome alignment and used it for phylogenetic and coalescent analyses. We also screened the genomes to determine presence of antibiotic resistance conferring genes. RESULTS: Population structure analysis grouped the ST217 isolates into five sequence clusters (SCs), which were highly associated with different geographical regions and showed limited intracontinental and intercontinental spread. The SCs showed lower than expected genomic sequence, which suggested strong purifying selection and small population sizes caused by bottlenecks. Recombination rates varied between the SCs but were lower than in other successful clones such as PMEN1. African isolates showed higher prevalence of antibiotic resistance genes than Asian isolates. Interestingly, certain West African isolates harbored a defective chloramphenicol and tetracycline resistance-conferring element (Tn5253) with a deletion in the loci encoding the chloramphenicol resistance gene (cat pC194), which caused lower chloramphenicol than tetracycline resistance. Furthermore, certain genes that promote colonisation were absent in the isolates, which may contribute to serotype 1's rarity in carriage and consequently its lower recombination rates. CONCLUSIONS: The high phylogeographic diversity of the ST217 clone shows that this clone has been in circulation globally for a long time, which allowed its diversification and adaptation in different geographical regions. Such geographic adaptation reflects local variations in selection pressures in different locales. Further studies will be required to fully understand the biological mechanisms which makes the ST217 clone highly invasive but unable to successfully colonise the human nasopharynx for long durations which results in lower recombination rates.

Recombination in Streptococcus pneumoniae Lineages Increase with Carriage Duration and Size of the Polysaccharide Capsule.

Streptococcus pneumoniae causes a high burden of invasive pneumococcal disease (IPD) globally, especially in children from resource-poor settings. Like many bacteria, the pneumococcus can import DNA from other strains or even species by transformation and homologous recombination, which has allowed the pneumococcus to evade clinical interventions such as antibiotics and pneumococcal conjugate vaccines (PCVs). Pneumococci are enclosed in a complex polysaccharide capsule that determines the serotype; the capsule varies in size and is associated with properties including carriage prevalence and virulence. We determined and quantified the association between capsule and recombination events using genomic data from a diverse collection of serotypes sampled in Malawi. We determined both the amount of variation introduced by recombination relative to mutation (the relative rate) and how many individual recombination events occur per isolate (the frequency). Using univariate analyses, we found an association between both recombination measures and multiple factors associated with the capsule, including duration and prevalence of carriage. Because many capsular factors are correlated, we used multivariate analysis to correct for collinearity. Capsule size and carriage duration remained positively associated with recombination, although with a reduced P value, and this effect may be mediated through some unassayed additional property associated with larger capsules. This work describes an important impact of serotype on recombination that has been previously overlooked. While the details of how this effect is achieved remain to be determined, it may have important consequences for the serotype-specific response to vaccines and other interventions. IMPORTANCE: The capsule determines >90 different pneumococcal serotypes, which vary in capsule size, virulence, duration, and prevalence of carriage. Current serotype-specific vaccines elicit anticapsule antibodies. Pneumococcus can take up exogenous DNA by transformation and insert it into its chromosome by homologous recombination. This mechanism has disseminated drug resistance and generated vaccine escape variants. It is hence crucial to pneumococcal evolutionary response to interventions, but there has been no systematic study quantifying whether serotypes vary in recombination and whether this is associated with serotype-specific properties such as capsule size or carriage duration. Larger capsules could physically inhibit DNA uptake, or given the longer carriage duration for larger capsules, this may promote recombination. We find that recombination varies among capsules and is associated with capsule size, carriage duration, and carriage prevalence and negatively associated with invasiveness. The consequence of this work is that serotypes with different capsules may respond differently to selective pressures like vaccines.

Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice.

Gram-positive bacterial pathogens that secrete cytotoxic pore-forming toxins, such as Staphylococcus aureus and Streptococcus pneumoniae, cause a substantial burden of disease. Inspired by the principles that govern natural toxin-host interactions, we have engineered artificial liposomes that are tailored to effectively compete with host cells for toxin binding. Liposome-bound toxins are unable to lyse mammalian cells in vitro. We use these artificial liposomes as decoy targets to sequester bacterial toxins that are produced during active infection in vivo. Administration of artificial liposomes within 10 h after infection rescues mice from septicemia caused by S. aureus and S. pneumoniae, whereas untreated mice die within 24-33 h. Furthermore, liposomes protect mice against invasive pneumococcal pneumonia. Composed exclusively of naturally occurring lipids, tailored liposomes are not bactericidal and could be used therapeutically either alone or in conjunction with antibiotics to combat bacterial infections and to minimize toxin-induced tissue damage that occurs during bacterial clearance.

The B lymphocyte differentiation factor (BAFF) is expressed in the airways of children with CF and in lungs of mice infected with Pseudomonas aeruginosa.

BACKGROUND: Chronic lung infection with Pseudomonas aeruginosa remains a major cause of mortality and morbidity among individuals with CF. Expression of mediators promoting recruitment and differentiation of B cells, or supporting antibody production is poorly understood yet could be key to controlling infection. METHODS: BAFF was measured in BAL from children with CF, both with and without P. aeruginosa, and controls. Mice were intra-nasally infected with P. aeruginosa strain LESB65 for up to 7 days. Cellular infiltration and expression of B cell chemoattractants and B cell differentiation factor, BAFF were measured in lung tissue. RESULTS: BAFF expression was elevated in both P. aeruginosa negative and positive CF patients and in P. aeruginosa infected mice post infection. Expression of the B cell chemoattractants CXCL13, CCL19 and CCL21 increased progressively post infection. CONCLUSIONS: In a mouse model, infection with P. aeruginosa was associated with elevated expression of BAFF and other B cell chemoattractants suggesting a role for airway B cell recruitment and differentiation in the local adaptive immune response to P. aeruginosa. The paediatric CF airway, irrespective of pseudomonal infection, was found to be associated with an elevated level of BAFF implying that BAFF expression is not specific to pseudomonas infection and may be a feature of the CF airway. Despite the observed presence of a potent B cell activator, chronic colonisation is common suggesting that this response is ineffective.

The AgI/II family adhesin AspA is required for respiratory infection by Streptococcus pyogenes.

Streptococcus pyogenes (GAS) is a human pathogen that causes pharyngitis and invasive diseases such as toxic shock syndrome and sepsis. The upper respiratory tract is the primary reservoir from which GAS can infect new hosts and cause disease. The factors involved in colonisation are incompletely known however. Previous evidence in oral streptococci has shown that the AgI/II family proteins are involved. We hypothesized that the AspA member of this family might be involved in GAS colonization. We describe a novel mouse model of GAS colonization of the nasopharynx and lower respiratory tract to elucidate these interactions. We used two clinical M serotypes expressing AspA, and their aspA gene deletant isogenic mutants in experiments using adherence assays to respiratory epithelium, macrophage phagocytosis and neutrophil killing assays and in vivo models of respiratory tract colonisation and infection. We demonstrated the requirement for AspA in colonization of the respiratory tract. AspA mutants were cleared from the respiratory tract and were deficient in adherence to epithelial cells, and susceptible to phagocytosis. Expression of AspA in the surrogate host Lactococcus lactis protected bacteria from phagocytosis. Our results suggest that AspA has an essential role in respiratory infection, and may function as a novel anti-phagocytic factor.

Controlled human infection and rechallenge with Streptococcus pneumoniae reveals the protective efficacy of carriage in healthy adults.

RATIONALE: The immunological and protective role of pneumococcal carriage in healthy adults is not known, but high rates of disease and death in the elderly are associated with low carriage prevalence. OBJECTIVES: We employed an experimental human pneumococcal carriage model to investigate the immunizing effect of a single carriage episode. METHODS: Seventy healthy adults were challenged, and of those with carriage, 10 were rechallenged intranasally with live 6B Streptococcus pneumoniae up to 11 months after clearance of the first carriage episode. Serum and nasal wash antibody responses were measured before and after each challenge. MEASUREMENTS AND MAIN RESULTS: A total of 29 subjects were experimentally colonized. No subjects were colonized by experimental rechallenge, demonstrating the protective effect of initial carriage against subsequent infection. Carriage increased both mucosal and serum IgG levels to pneumococcal proteins and polysaccharide, resulting in a fourfold increase in opsonophagocytic activity. Importantly, passive transfer of postcarriage sera from colonized subjects conferred 70% protection against lethal challenge by a heterologous strain in a murine model of invasive pneumococcal pneumonia. These levels were significantly higher than the protection conferred by either precarriage sera (30%) or saline (10%). CONCLUSIONS: Experimental human carriage resulted in mucosal and systemic immunological responses that conferred protection against recolonization and invasive pneumococcal disease. These data suggest that mucosal pneumococcal vaccination strategies may be important for vulnerable patient groups, particularly the elderly, who do not sustain carriage.

P4-mediated antibody therapy in an acute model of invasive pneumococcal disease.

New treatments against severe bacterial infections are needed because the response to antibiotic treatment is slow in acute settings and is becoming less effective owing to the emergence of antibiotic-resistant pathogens. P4-mediated antibody therapy offers a unique treatment strategy that combines exogenous immunoglobulin with the immunoactivating peptide P4. In an acute model of pneumococcal disease, mice were infected with Streptococcus pneumoniae and treated intravenously or intranasally with P4 and intravenous immunoglobulin (IVIG). Survival of P4-IVIG-treated mice increased from 0% to 60% among those that received intravenous treatment and from 0% to 100% among those that received intranasal treatment. Importantly, intranasal administration of P4 at an early stage of infection prevented the onset of bacteremia and sepsis. Increased survival was associated with reduced bacterial burden in affected tissues and with recruitment and activation of professional phagocytes, as manifested by increased expression of Fc-γ receptors. In vitro studies involving P4-stimulated alveolar, peritoneal, and J774.2 murine macrophages showed an increased ability of these immune cells to phagocytose pneumococci independent of capsule. The use of adjunct antibody therapies to treat infectious diseases shows promise.