Interferon-γ promotes monocyte-mediated lung injury during influenza infection.

Influenza A virus (IAV) infection triggers an exuberant host response that promotes acute lung injury. However, the host response factors that promote the development of a pathologic inflammatory response to IAV remain incompletely understood. In this study, we identify an interferon-γ (IFN-γ)-regulated subset of monocytes, CCR2+ monocytes, as a driver of lung damage during IAV infection. IFN-γ regulates the recruitment and inflammatory phenotype of CCR2+ monocytes, and mice deficient in CCR2 (CCR2-/-) or IFN-γ (IFN-γ-/-) exhibit reduced lung inflammation, pathology, and disease severity. Adoptive transfer of wild-type (WT) (IFN-γR1+/+) but not IFN-γR1-/- CCR2+ monocytes restore the WT-like pathological phenotype of lung damage in IAV-infected CCR2-/- mice. CD8+ T cells are the main source of IFN-γ in IAV-infected lungs. Collectively, our data highlight the requirement of IFN-γ signaling in the regulation of CCR2+ monocyte-mediated lung pathology during IAV infection.

An Account of Immune Senescence in the Clinical Pathophysiology of COVID-19 Infection in Aging.

Worldwide COVID-19 infection poses an enormous risk to public health and an alarming global socioeconomic burden. The impact of the COVID-19 pandemic on individuals with underlying health conditions as well as on the elderly population is extensive and effective strategies are needed to understand the mechanism behind it. Cellular senescence defines as an irreversible cell cycle arrest due to DNA damage leading to accumulation of senescent cells in the elderly population and may result in worsening of COVID-19 mediated increased mortality. However, whether this variation in senescence levels, in different aged populations, translation to COVID-19 infection is unknown. The spike protein of SARS-CoV-2 has been recently identified to be responsible for inducing pathogenic signals, although a clear understanding of how the host receptor interacts with SARS-CoV-2 protein and mediates the immune responses is not clear. In this review, we address the epidemiology of SARS-CoV-2 and the cellular senescence responding immune response to pathogenic SARS-CoV-2. We provide a prospective summary of what to expect and how to brace the possible immunological strategy to protect against COVID-19 infection. The review majorly explores an underline mechanism of how senescent cells trigger a hyperimmune inflammatory response and cause high mortality in aging people could serve as a potential aid to alleviate the treatment for elderly battling COVID-19 infection.

Androgen receptor activation alleviates airway hyperresponsiveness, inflammation, and remodeling in a murine model of asthma.

Epidemiological studies demonstrate an apparent sex-based difference in the prevalence of asthma, with a higher risk in boys than girls, which is reversed postpuberty, where women become more prone to asthma than men, suggesting a plausible beneficial role for male hormones, especially androgens as a regulator of pathophysiology in asthmatic lungs. Using a murine model of asthma developed with mixed allergen (MA) challenge, we report a significant change in airway hyperresponsiveness (AHR), as demonstrated by increased thickness of epithelial and airway smooth muscle layers and collagen deposition, as well as Th2/Th17-biased inflammation in the airways of non-gonadectomized (non-GDX) and gonadectomized (GDX) male mice. Here, compared with non-GDX mice, MA-induced AHR and inflammatory changes were more prominent in GDX mice. Activation of androgen receptor (AR) using 5α-dihydrotestosterone (5α-DHT, AR agonist) resulted in decreased Th2/Th17 inflammation and remodeling-associated changes, resulting in improved lung function compared with MA alone challenged mice, especially in GDX mice. These changes were not observed with Flutamide (Flut, AR antagonist). Overall, we show that AR exerts a significant and beneficial role in asthma by regulating AHR and inflammation.

An Overview of Flow Cytometry: Its Principles and Applications in Allergic Disease Research.

Flow cytometry is a popular technique used for both clinical and research purposes. It involves laser-based technology to characterize cells based on size, shape, and complexity. Additionally, flow cytometers are equipped with the ability to take fluorescence measurements at multiple wavelengths. This capability makes the flow cytometer a practical resource in the utilization of fluorescently conjugated antibodies, fluorescent proteins, DNA binding dyes, viability dyes, and ion indicator dyes. As the technology advances, the number of parameters a flow cytometer can measure has increased tremendously, and now some has the capacity to analyze 30-50 or more parameters on a single cell. Here, we describe the basic principles involved in the mechanics and procedures of flow cytometry along with an insight into applications of flow cytometry techniques for biomedical and allergic disease research.

The Application of Flow Cytometry for Simultaneous and Multi-parametric Analysis of Heterogenous Cell Populations in Basic and Clinical Research.

The use of flow cytometry allows simultaneous measurement and multiparametric analysis of single cells in a heterogenous solution. The purpose of flow cytometry can vary depending on the use of antibodies and dyes targeted for specific cell molecules. The method of immune-phenotyping with fluorescently conjugated antibodies to label cell proteins or DNA works in tandem with fluidic, optic, and electrical systems present in the flow cytometer. Some flow cytometers can detect numerous fluorescent molecules on a single cell, allowing the measurement of more than 30 parameters. This ability to detect, measure, and quantitate multiple fluorescent markers on a single cell makes the flow cytometer a useful tool for analyzing various aspects of cell phenotype and function. Here we describe a standardized protocol for surface and intracellular immune-phenotyping of murine lungs, beginning with the building of an optimal antibody panel and ending with data analysis and representation, including sample gating strategies for innate and adaptive immune responses.

IL-6 Deficiency Exacerbates Allergic Asthma and Abrogates the Protective Effect of Allergic Inflammation against Streptococcus pneumoniae Pathogenesis.

Allergic asthma (AA) is characterized as a Th2-biased airway inflammation that can develop lung inflammation and remodeling of the respiratory tract. Streptococcus pneumoniae is a major respiratory pathogen, causing noninvasive (otitis media and pneumonia) and invasive diseases (sepsis) in humans. We sought to determine the role of IL-6 in the regulation of lung inflammation in murine AA caused by Aspergillus fumigatus as well as its consequence on the regulation of airway barrier integrity and S. pneumoniae disease. In an AA model, IL-6 deficiency led to increased lung inflammation, eosinophil recruitment, tissue pathology, and collagen deposition. Additionally, IL-6-deficient asthmatic mice exhibited reduced goblet cell hyperplasia and increased TGF-ß production. These key changes in the lungs of IL-6-deficient asthmatic mice resulted in dysregulated tight junction proteins and increased lung permeability. Whereas the host response to AA protected against S. pneumoniae lung disease, the IL-6 deficiency abrogated the protective effect of allergic inflammation against S. pneumoniae pathogenesis. Consistent with in vivo data, IL-6 knockdown by small interfering RNA or the blockade of IL-6R signaling exacerbated the TGF-ß-induced dysregulation of tight junction proteins, E-cadherin and N-cadherin expression, and STAT3 phosphorylation in MLE-12 epithelial cells. Our findings demonstrate a previously unrecognized role of host IL-6 response in the regulation of lung inflammation during AA and the control of S. pneumoniae bacterial disease. A better understanding of the interactions between lung inflammation and barrier framework could lead to the development of therapies to control asthma inflammation and preserve barrier integrity.

Double-Edged Role of Interleukin 17A in Streptococcus pneumoniae Pathogenesis During Influenza Virus Coinfection.

BACKGROUND: We sought to determine the role of host interleukin 17A (IL-17A) response against colonizing Streptococcus pneumoniae, and its transition to a pathogen during coinfection with an influenza virus, influenza A H1N1 A/Puerto Rico/8/1934 (PR8). METHOD: Wild-type (WT) C57BL/6 mice were intranasally inoculated with S. pneumoniae serotype 6A to establish colonization and later infected with the influenza strain, PR8, resulting in invasive S. pneumoniae disease. The role of the IL-17A response in colonization and coinfection was investigated in WT, RoRγt-/- and RAG1-/- mice with antibody-mediated depletion of IL-17A (WT) and CD90 cells (RAG1-/-). RESULTS: RAG1-/- mice did not clear colonization and IL-17A neutralization impaired 6A clearance in WT mice. RoRγt-/- mice also had reduced clearance. S. pneumoniae-PR8 coinfection elicited a robust IL-17A response in the nasopharynx; IL-17A neutralization reduced S. pneumoniae invasive disease. RoRγt-/- mice also had reduced S. pneumoniae disease in a coinfection model. Depletion of CD90+ cells suppressed the IL-17A response and reduced S. pneumoniae invasion in RAG1-/- mice. CONCLUSION: Our data show that although IL-17A reduces S. pneumoniae colonization, coinfection with influenza virus elicits a robust innate IL-17A response that promotes inflammation and S. pneumoniae disease in the nasopharynx.

Role of Inflammatory Risk Factors in the Pathogenesis of Streptococcus pneumoniae.

Streptococcus pneumoniae (Spn) is a colonizer of the human nasopharynx (NP), causing a variety of infections in humans including otitis media, pneumonia, sepsis, and meningitis. The NP is an immune permissive site which allows for the persistence of commensal bacteria. Acute or chronic respiratory airway inflammation constitutes a significant risk factor for the manifestation of Spn infections. The inflammatory conditions caused by an upper respiratory viral infection or respiratory conditions such as allergic asthma and chronic obstructive pulmonary disorders (COPDs) are implicated in the dysregulation of airway inflammation and tissue damage, which compromise the respiratory barrier integrity. These immune events promote bacterial outgrowth leading to Spn dissemination and invasion into the bloodstream. Therefore, suppression of inflammation and restoration of respiratory barrier integrity could contain Spn infections manifesting in the backdrop of an inflammatory disease condition. The gained knowledge could be harnessed in the design of novel therapeutic interventions to circumvent Spn bacterial infections.

Immunogenicity and protective efficacy of monovalent PCVs containing 22F and 33F polysaccharides in mouse models of colonization and co-infection.

BACKGROUND: In the current transmission, we studied the immunogenicity and protective efficacy of serotypes 22F and 33F in the prevention of colonization and of invasive Streptococcus pneumoniae (Spn) pathogenesis during an influenza co-infection. Serotypes 22F and 33F are emerging Spn serotypes, which are not part of currently administered pneumococcal conjugate vaccine formulations (PCVs). Spn serotype 6A is an ingredient in the currently administered PCV13 vaccine and was therefore included in the study as a control. METHODS: Adult (six weeks) and infant (two weeks) C57BL/6 mice were intranasally infected in the nasopharynx (NP) with Spn serotypes 22F, 33F, or 6A. Influenza A H1N1 A/Puerto Rico/8/193 virus (PR8) was introduced one day after the NP Spn colonization. In an immunization challenge study, mice were vaccinated with monovalent 22F, 33F, or 6A polysaccharide conjugated to the CRM197 antigen. The immunized mice were colonized or co-infected to study the vaccines efficacy. RESULTS: All three Spn serotypes established colonization in adult and infant mice. The co-infected mice showed an increase in Spn NP density. Invasive Spn infection (bacteremia) was observed following the co-infection with serotypes 22F and 6A but not 33F in adult mice, whereas infant mice developed bacteremia following co-infection with all three Spn serotypes. The vaccinations led to robust serum antibody responses to capsular polysaccharides 22F, 6A, and less for 33F. The vaccinations resulted in reductions of Spn NP colonization density for all three serotypes, prevention of bacteremia, and increased survival with Spn serotypes 22F and 6A. Passive transfer of antisera was associated with a reduction of Spn colonization densities in infant mice. CONCLUSION: Vaccinations with monovalent 22F, 33F, or 6A formulations protect against Spn colonization, and the efficacy of the 22F vaccination was comparable to the 6A vaccination in preventing an invasive Spn bacterial infection during an influenza co-infection.

Intranasal coinfection model allows for assessment of protein vaccines against nontypeable Haemophilus influenzae in mice.

PURPOSE: Nontypeable Haemophilus influenzae (NTHi) is a commensal in the human nasopharynx and the cause of pneumonia, meningitis, sinusitis, acute exacerbations of chronic obstructive pulmonary disease and acute otitis media (AOM). AOM is the most common ailment for which antibiotics are prescribed in the United States. With the emergence of new strains of antibiotic-resistant bacteria, finding an effective and broad coverage vaccine to protect against AOM-causing pathogens has become a priority. Mouse models are a cost-effective and efficient way to help determine vaccine efficacy. Here, we describe an NTHi AOM model in C57BL/6J mice, which also utilizes a mouse-adapted H1N1 influenza virus to mimic human coinfection. METHODOLOGY: We tested our coinfection model using a protein vaccine formulation containing protein D, a well-studied NTHi vaccine candidate that can be found in the 10-valent Streptococcus pneumoniae conjugate vaccine. We verified the usefulness of our mouse model by comparing bacterial loads in the nose and ear between protein D-vaccinated and control mice. RESULTS: While there was no measurable difference in nasal bacterial loads, we did detect significant differences in the bacterial loads of ear washes and ear bullae between vaccinated and control mice. CONCLUSION: The results from this study suggest that our NTHi AOM coinfection model is useful for assessing protein vaccines.

Protection against Streptococcus pneumoniae Invasive Pathogenesis by a Protein-Based Vaccine Is Achieved by Suppression of Nasopharyngeal Bacterial Density during Influenza A Virus Coinfection.

An increase in Streptococcus pneumoniae nasopharynx (NP) colonization density during a viral coinfection initiates pathogenesis. To mimic natural S. pneumoniae pathogenesis, we commensally colonized the NPs of adult C57BL/6 mice with S. pneumoniae serotype (ST) 6A or 8 and then coinfected them with mouse-adapted H1N1 influenza A virus (PR/8/34). S. pneumoniae established effective commensal colonization, and influenza virus coinfection caused S. pneumoniae NP density to increase, resulting in bacteremia and mortality. We then studied histidine triad protein D (PhtD), an S. pneumoniae adhesin vaccine candidate, for its ability to prevent invasive S. pneumoniae disease in adult and infant mice. In adult mice, the efficacy of PhtD vaccination was compared with that of PCV13. Vaccination with PCV13 led to a greater reduction of S. pneumoniae NP density (>2.5 log units) than PhtD vaccination (∼1-log-unit reduction). However, no significant difference was observed with regard to the prevention of S. pneumoniae bacteremia, and there was no difference in mortality. Depletion of CD4+ T cells in PhtD-vaccinated adult mice, but not PCV13-vaccinated mice, caused a loss of vaccine-induced protection. In infant mice, passive transfer of antisera or CD4+ T cells from PhtD-vaccinated adult mice led to a nonsignificant reduction in NP colonization density, whereas passive transfer of antisera and CD4+ T cells was needed to cause a significant reduction in NP colonization density. For the first time, these data show an outcome with regard to prevention of invasive S. pneumoniae pathogenesis with a protein vaccine similar to that which occurs with a glycoconjugate vaccine despite a less robust reduction in NP bacterial density.

Developing a vaccine to prevent otitis media caused by nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is a predominant organism of the upper respiratory nasopharyngeal microbiota. Its disease spectrum includes otitis media, sinusitis, non-bacteremic pneumonia and invasive infections. Protein-based vaccines to prevent NTHi infections are needed to alleviate these infections in children and vulnerable populations such as the elderly and those with chronic obstructive pulmonary disease (COPD). One NTHi protein is included in a pneumococcal conjugate vaccine and has been shown to provide efficacy. Our lab has been interested in understanding the immunogenicity of NTHi vaccine candidates P6, protein D and OMP26 for preventing acute otitis media in young children. We expect that continued investigation and progress in the development of an efficacious protein based vaccine against NTHi infections is achievable in the near future.

Next generation protein based Streptococcus pneumoniae vaccines.

All currently available Streptococcus pneumoniae (Spn) vaccines have limitations due to their capsular serotype composition. Both the 23-valent Spn polysaccharide vaccine (PPV) and 7, 10, or 13-valent Spn conjugate vaccines (PCV-7, 10, -13) are serotype-based vaccines and therefore they elicit only serotype-specific immunity. Emergence of replacement Spn strains expressing other serotypes has consistently occurred following introduction of capsular serotype based Spn vaccines. Furthermore, capsular polysaccharide vaccines are less effective in protection against non-bacteremic pneumonia and acute otitis media (AOM) than against invasive pneumococcal disease (IPD). These shortcomings of capsular polysaccharide-based Spn vaccines have created high interest in development of non-serotype specific protein-based vaccines that could be effective in preventing both IPD and non-IPD infections. This review discusses the progress to date on development of Spn protein vaccine candidates that are highly conserved by all Spn strains, are highly conserved, exhibit maximal antigenicity and minimal reactogenicity to replace or complement the current capsule-based vaccines. Key to development of a protein based Spn vaccine is an understanding of Spn pathogenesis. Based on pathogenesis, a protein-based Spn vaccine should include one or more ingredients that reduce NP colonization below a pathogenic inoculum. Elimination of all Spn colonization may not be achievable or even advisable. The level of expression of a target protein antigen during pathogenesis is another key to the success of protein based vaccines.. As with virtually all currently licensed vaccines, production of a serum antibody response in response to protein based vaccines is anticipated to provide protection from Spn infections. A significant advantage that protein vaccine formulations can offer over capsule based vaccination is their potential benefits associated with natural priming and boosting to all strains of Spn. One of the most universal and comprehensive approaches of identifying novel vaccine candidates is the investigation of human sera from different disease stages of natural infections. Antigens that are robustly reactive in preliminary human serum screening constitute a pathogen-specific antigenome. This strategy has identified a number of Spn protein vaccine candidates that are moving forward in human clinical trials.

An ahemolytic pneumolysin of Streptococcus pneumoniae manipulates human innate and CD4⁺ T-cell responses and reduces resistance to colonization in mice in a serotype-independent manner.

BACKGROUND: Some Streptococcus pneumoniae serotypes express an ahemolytic pneumolysin (PLYa). Serotypes that commonly express PLYa, including serotype 8 (ST8) and ST1, are often associated with a low prevalence during colonization but a higher propensity to cause invasive disease. We sought to study the host response to ST8 PLYa in a homologous and heterologous capsular background. METHODS: We genetically exchanged the PLYa of ST8 strain 6308 with the hemolytic PLY (PLYh) of ST3 A66.1 and vice versa and determined the impact of the exchange on nasopharyngeal colonization in mice. Then, to compare the response of human cells to PLYa-expressing and PLYh-expressing strains, we infected human peripheral blood mononuclear cells (PBMCs) with PLY-switched strains and assessed dendritic cell and CD4(+) T-cell responses by intracellular cytokine staining. RESULT: Mice colonized with PLYa-expressing strains had significantly higher colonization densities than those colonized with PLYh-expressing strains, irrespective of capsular background. Compared with infection of PBMCs with PLYh-expressing strains, infection with PLYa-expressing strains induced diminished innate (dendritic cell cytokines, costimulatory receptor, and apoptotic) and adaptive (CD4(+) T-cell proliferative and memory interleukin 17A) responses. CONCLUSION: Our findings demonstrate that PLYa has the potential to manipulate host immunity irrespective of capsule type. PLY exchange between STs expressing PLYa and PLYh could lead to unexpected colonization or invasion phenotypes.

The host immune dynamics of pneumococcal colonization: implications for novel vaccine development.

The human nasopharynx (NP) microbiota is complex and diverse and Streptococcus pneumoniae (pneumococcus) is a frequent member. In the first few years of life, children experience maturation of their immune system thereby conferring homeostatic balance in which pneumococci are typically rendered as harmless colonizers in the upper respiratory environment. Pneumococcal carriage declines in many children before they acquire capsular-specific antibodies, suggesting a capsule antibody-independent mechanism of natural protection against pneumococcal carriage in early childhood. A child's immune system in the first few years of life is Th2-skewed so as to avoid inflammation-induced immunopathology. Understanding Th1/Th2 and Th17 ontogeny in early life and how adjuvant vaccine formulations shift the balance of T helper-cell differentiation, may facilitate the development of new protein-based pneumococcal vaccines. This article will discuss the immune dynamics of pneumococcal colonization in infants. The discussion aims to benefit the design and improvement of protein subunit-based next-generation pneumococcal vaccines.

Dual orientation of the outer membrane lipoprotein P6 of nontypeable haemophilus influenzae.

The majority of outer membrane (OM) lipoproteins in Gram-negative bacteria are tethered to the membrane via an attached lipid moiety and oriented facing in toward the periplasmic space; a few lipoproteins have been shown to be surface exposed. The outer membrane lipoprotein P6 from the Gram-negative pathogenic bacterium nontypeable Haemophilus influenzae (NTHi) is surface exposed and a leading vaccine candidate for prevention of NTHi infections. However, we recently found that P6 is not a transmembrane protein as previously thought (L. V. Michel, B. Kalmeta, M. McCreary, J. Snyder, P. Craig, M. E. Pichichero, Vaccine 29:1624-1627, 2011). Here we pursued studies to show that P6 has a dual orientation, existing infrequently as surface exposed and predominantly as internally oriented toward the periplasmic space. Flow cytometry using three monoclonal antibodies with specificity for P6 showed surface staining of whole NTHi cells. Confocal microscopy imaging confirmed that antibodies targeted surface-exposed P6 of intact NTHi cells and not internal P6 in membrane-compromised or dead cells. Western blots of two wild-type NTHi strains and a mutant NTHi strain that does not express P6 showed that P6 antibodies do not detect a promiscuous epitope on NTHi. Depletion of targets to nonlipidated P6 significantly decreased bactericidal activity of human serum. Protease digestion of surface-exposed P6 demonstrated that P6 is predominantly internally localized in a manner similar to its homologue Pal in Escherichia coli. We conclude that P6 of NTHi is likely inserted into the OM in two distinct orientations, with the predominant orientation facing in toward the periplasm.

PcpA of Streptococcus pneumoniae mediates adherence to nasopharyngeal and lung epithelial cells and elicits functional antibodies in humans.

Streptococcus pneumoniae (pneumococci) adhere to human nasopharyngeal (NP) epithelial cells as a first step in colonization and adherence of pneumococci to lung epithelia may be required to establish pneumonia. We sought to determine if PcpA can serve as an adhesin to human NP (D562) and lung (A549) epithelial cells and whether PcpA mediated adherence can be inhibited by human anti-PcpA antibodies. A PcpA isogenic mutant was constructed in a pneumococcal TIGR4 background. When the mutant and wild type strains were compared for their adherence to D562 and A549 cell lines, a reduction in adherence by the mutant was observed (p = 0.0001 for both cell types). PcpA was ectopically expressed on the surface of minimally-adherent heterologous host Escherichia coli resulting in augmented adherence to D562 (p = 0.002) and A549 (p = 0.015) cells. Total IgG was purified from a pool of 6 human sera having high IgG titers of anti-pneumococcal proteins. The purified IgG reduced TIGR4 adherence to D562 cells but we determined that this effect was largely due to bacterial cell aggregation as determined by flow cytometry and confocal microscopy. Fab fragments were prepared from pooled IgG sera. Inhibition of TIGR4 adherence to D562 cells was observed using the Fab fragments without causing bacterial aggregation (p = 0.0001). Depletion of PcpA-specific Fab fragments resulted in an increase in adherence of TIGR4 to D562 cells (p = 0.028). We conclude that PcpA can mediate adherence of pneumococci to human NP and lung epithelial cells and PcpA mediated adherence can be inhibited by human anti-PcpA antibodies.

Bactericidal antibody response against P6, protein D, and OMP26 of nontypeable Haemophilus influenzae after acute otitis media in otitis-prone children.

The bactericidal antibody response to three nontypeable Haemophilus influenzae (NTHi) outer membrane proteins (D, P6, and OMP26) was studied in 24 otitis-prone children (aged 7-28 months) after an acute otitis media (AOM) caused by NTHi. The study was carried out to understand the contribution of antigen-specific bactericidal antibody responses in the class of children who are most vulnerable to recurrent otitis media infections. Levels of protein D (P = 0.005) and P6 (P = 0.026) but not OMP26 antibodies were higher in bactericidal sera compared with nonbactericidal sera. For five (24%) and 16 (76%) of 21 bactericidal sera tested, removal of anti-protein D and P6 antibody, respectively, resulted in a two- to fourfold drop in bactericidal antibody. Antibodies to OMP26 did not make any contribution to the overall bactericidal activity in any serum samples. Eleven of 21 sera (52%) had bactericidal activity against a heterologous NTHi (86-028 NP) strain but the titers were significantly lower (P < 0.05) as compared to the homologous strains. Future studies of protein D, P6, OMP26, and other potential NTHi vaccine antigens should include studies of bactericidal antibody in children who are otitis prone as a possible correlate of protection.

Vaccine candidates PhtD and PhtE of Streptococcus pneumoniae are adhesins that elicit functional antibodies in humans.

We evaluated the role of vaccine candidate surface proteins, PhtD and PhtE as antigens with functional importance for Streptococcus pneumoniae (pneumococci) in adherence to nasopharyngeal (D562) and lung (A549) epithelial cell lines. Comparing TIGR4 to PhtD and PhtE- isogenic mutants, a 40% (p=0.001) and 42% (p=0.002) drop in the number of epithelial cells with adherent pneumococci was observed to both cells lines with the mutants, as quantitated using flow cytometry. We expressed PhtD and PhtE on the surface of Escherichia coli and demonstrated that when PhtD and PhtE were surface expressed on E. coli, adherence increased to D562 and A549 cells, compared with the E. coli parent strain (p=0.005, 0.013 for D562 and p=0.034, p=0.035 for A549). Using flow cytometry and confocal microscopy we found that pneumococci aggregated in the presence of human serum IgG, leading to a non-specific drop in adherence. Therefore IgG Fab fragments were prepared to study the functional role of PhtD and PhtE-specific Fabs in blocking adherence. The addition of 1µg of IgG Fab from adult sera led to a 34% reduction (p=0.002) and from children a 20% (p=0.023) reduction in D562 epithelial cells with adherent pneumococci. In purified IgG from adult sera, the depletion of PhtD and PhtE specific Fab from total IgG Fab resulted in a significant increase in the number of D562 epithelial cells with adherent pneumococci (p=0.005 for PhtD and p=0.024 for PhtE). We conclude that antibody directed to PhtD and PhtE adhesins of pneumococci, if raised by vaccination, may function to prevent pneumococcal adherence to human airway epithelial cells.