Elena Mitsi

First Human Challenge Testing of a Pneumococcal Vaccine. Double-Blind Randomized Controlled Trial.

RATIONALE New vaccines are urgently needed to protect the vulnerable from bacterial pneumonia. Clinical trials of pneumonia vaccines are slow and costly, requiring tens of thousands of patients. Studies of pneumococcal vaccine efficacy against colonization have been proposed as a novel method to down-select between vaccine candidates. OBJECTIVES Using our safe and reproducible experimental human pneumococcal colonization model, we aimed to determine the effect of 13-valent pneumococcal conjugate vaccine (PCV) on colonization. METHODS A total of 100 healthy participants aged 18-50 years were recruited into this double-blind randomized placebo-controlled trial. They were randomly assigned to PCV (n = 49) or hepatitis A (control, n = 50) vaccination and inoculated with 80,000 CFU/100 μl of Streptococcus pneumoniae (6B) per naris. MEASUREMENTS AND MAIN RESULTS Participants were followed up for 21 days to determine pneumococcal colonization by culture of nasal wash. The PCV group had a significantly reduced rate of 6B colonization (10% [5 of 48]) compared with control subjects (48% [23 of 48]) (risk ratio, 0.22; confidence interval, 0.09-0.52; P < 0.001). Density of colonization was reduced in the PCV group compared with the control group following inoculation. The area under the curve (density vs. day) was significantly reduced in the PCV compared with control group (geometric mean, 259 vs. 11,183; P = 0.017). CONCLUSIONS PCV reduced pneumococcal colonization rate, density, and duration in healthy adults. The experimental human pneumococcal colonization model is a safe, cost-effective, and efficient method to determine the protective efficacy of new vaccines on pneumococcal colonization; PCV provides a gold standard against which to test these novel vaccines. Clinical trial registered with ISRCTN 45340436.

Agglutination by anti-capsular polysaccharide antibody is associated with protection against experimental human pneumococcal carriage.

The ability of pneumococcal conjugate vaccine (PCV) to decrease transmission by blocking the acquisition of colonization has been attributed to herd immunity. We describe the role of mucosal immunoglobulin G (IgG) to capsular polysaccharide (CPS) in mediating protection from carriage, translating our findings from a murine model to humans. We used a flow cytometric assay to quantify antibody-mediated agglutination demonstrating that hyperimmune sera generated against an unencapsulated mutant was poorly agglutinating. Passive immunization with this antiserum was ineffective to block acquisition of colonization compared to agglutinating antisera raised against the encapsulated parent strain. In the human challenge model, samples were collected from PCV and control-vaccinated adults. In PCV-vaccinated subjects, IgG levels to CPS were increased in serum and nasal wash (NW). IgG to the inoculated strain CPS dropped in NW samples after inoculation suggesting its sequestration by colonizing pneumococci. In post-vaccination NW samples pneumococci were heavily agglutinated compared with pre-vaccination samples in subjects protected against carriage. Our results indicate that pneumococcal agglutination mediated by CPS-specific antibodies is a key mechanism of protection against acquisition of carriage. Capsule may be the only vaccine target that can elicit strong agglutinating antibody responses, leading to protection against carriage acquisition and generation of herd immunity.

Augmented Passive Immunotherapy with P4 Peptide Improves Phagocyte Activity in Severe Sepsis

Introduction: Antimicrobial resistance threatens to undermine treatment of severe infection; new therapeutic strategies are urgently needed. Preclinical work shows that augmented passive immunotherapy with P4 peptide increases phagocytic activity and shows promise as a novel therapeutic strategy. Our aim was to determine ex vivo P4 activity in a target population of patients admitted to critical care with severe infection. Methods: We prospectively recruited UK critical care unit patients with severe sepsis and observed clinical course (≥3 months postdischarge). Blood samples were taken in early (⩽48 h postdiagnosis, n = 54), latent (7 days postdiagnosis, n = 39), and convalescent (3–6 months postdiagnosis, n = 18) phases of disease. The primary outcome measure was killing of opsonized Streptococcus pneumoniae by neutrophils with and without P4 peptide stimulation. We also used a flow cytometric whole blood phagocytosis assay to determine phagocyte association and oxidation of intraphagosomal reporter beads. Results: P4 peptide increased neutrophil killing of opsonized pneumococci by 8.6% (confidence interval 6.35–10.76, P < 0.001) in all phases of sepsis, independent of infection source and microbiological status. This represented a 54.9% increase in bacterial killing compared with unstimulated neutrophils (15.6%) in early phase samples. Similarly, P4 peptide treatment significantly increased neutrophil and monocyte intraphagosomal reporter bead association and oxidation, independent of infection source. Conclusions: We have extended preclinical work to demonstrate that P4 peptide significantly increases phagocytosis and bacterial killing in samples from a target patient population with severe sepsis. This study supports the rationale for augmented passive immunotherapy as a therapeutic strategy in severe sepsis.

Effect of Live Attenuated Influenza Vaccine on Pneumococcal Carriage

The widely used nasally-administered Live Attenuated Influenza Vaccine (LAIV) alters the dynamics of naturally occurring nasopharyngeal carriage of Streptococcus pneumoniae in animal models. Using a human experimental model (serotype 6B) we tested two hypotheses: 1) LAIV increased the density of S. pneumoniae in those already colonised; 2) LAIV administration promoted colonisation. Randomised, blinded administration of LAIV or nasal placebo either preceded bacterial inoculation or followed it, separated by a 3-day interval. The presence and density of S. pneumoniae was determined from nasal washes by bacterial culture and PCR. Overall acquisition for bacterial carriage were not altered by prior LAIV administration vs. controls (25/55 [45.5%] vs 24/62 [38.7%] respectively, p=0.46). Transient increase in acquisition was detected in LAIV recipients at day 2 (33/55 [60.0%] vs 25/62 [40.3%] in controls, p=0.03). Bacterial carriage densities were increased approximately 10-fold by day 9 in the LAIV recipients (2.82 vs 1.81 log10 titers, p=0.03). When immunisation followed bacterial acquisition (n=163), LAIV did not change area under the bacterial density-time curve (AUC) at day 14 by conventional microbiology (primary endpoint), but significantly reduced AUC to day 27 by PCR (p=0.03). These studies suggest that LAIV may transiently increase nasopharyngeal density of S. pneumoniae. Transmission effects should therefore be considered in the timing design of vaccine schedules. Trial registration The study was registered on EudraCT (2014-004634-26) Funding The study was funded by the Bill and Melinda Gates Foundation and the UK Medical Research Council.

Human alveolar macrophages predominately express combined classical M1 and M2 surface markers in steady state

Alveolar macrophages (AM) are critical to the homeostasis of the inflammatory environment in the lung. Differential expression of surface markers classifies macrophages to either classically (M1) or alternatively activated (M2). We investigated the phenotype of human alveolar macrophages (AM) in adults living in two different geographical locations: UK and Malawi. We show that the majority of AM express high levels of M1 and M2 markers simultaneously, with the M1/M2 phenotype being stable in individuals from different geographical locations. The combined M1/M2 features confer to AM a hybrid phenotype, which does not fit the classic macrophage classification. This hybrid phenotype may confer to alveolar macrophages an ability to quickly switch between M1 or M2 associated functions allowing for appropriate responses to stimuli and tissue environment.

Assessing the ideal microwave duration for disinfection of sinus irrigation bottles- a quantitative study

Saline irrigation of the nasal cavity and paranasal sinuses has a recognised role in the management of chronic rhinosinusitis. However, bacterial recontamination of irrigation bottles through backflow from the sinonasal cavity is a concern in recurrent sinus cavity infections. While patients are encouraged to clean the irrigation bottles regularly, there remains significant concern that the use of contaminated bottles may perpetuate chronic rhinosinusitis. This study assesses the optimal microwave duration to achieve decontamination for each irrigation bottle component part (reservoir, tube and nozzle) using a standard, commercially available microwave. In addition, the irrigation fluid was also tested for contamination after each microwave cycle.

Inflammation induced by influenza virus impairs innate control of human pneumococcal carriage

Secondary bacterial pneumonia following influenza infection is a significant cause of mortality worldwide. Upper respiratory tract pneumococcal carriage is important as both determinants of disease and population transmission. The immunological mechanisms that contain pneumococcal carriage are well-studied in mice but remain unclear in humans. Loss of this control of carriage following influenza infection is associated with secondary bacterial pneumonia during seasonal and pandemic outbreaks. We used a human type 6B pneumococcal challenge model to show that carriage acquisition induces early degranulation of resident neutrophils and recruitment of monocytes to the nose. Monocyte function associated with clearance of pneumococcal carriage. Prior nasal infection with live attenuated influenza virus induced inflammation, impaired innate function and altered genome-wide nasal gene responses to pneumococcal carriage. Levels of the cytokine IP-10 promoted by viral infection at the time of pneumococcal encounter was positively associated with bacterial density. These findings provide novel insights in nasal immunity to pneumococcus and viral-bacterial interactions during co-infection.

Anti-protein immunoglobulin M responses to pneumococcus are not associated with aging

BackgroundThe incidence of community-acquired pneumonia and lower respiratory tract infection rises considerably in later life. Immunoglobulin M (IgM) antibody levels to pneumococcal capsular polysaccharide are known to decrease with age; however, whether levels of IgM antibody to pneumococcal proteins are subject to the same decline has not yet been investigated.MethodsThis study measured serum levels and binding capacity of IgM antibody specific to the pneumococcal surface protein A (PspA) and an unencapsulated pneumococcal strain in serum isolated from hospital patients aged < 60 and ≥ 60, with and without lower respiratory tract infection. A group of young healthy volunteers was used as a comparator to represent adults at very low risk of pneumococcal pneumonia. IgM serum antibody levels were measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry was performed to assess IgM binding capacity. Linear regression and one-way analysis of variance (ANOVA) tests were used to analyse the results.ResultsLevels and binding capacity of IgM antibody to PspA and the unencapsulated pneumococcal strain were unchanged with age.ConclusionsThese findings suggest that protein-based pneumococcal vaccines may provide protective immunity in the elderly.Trial registrationThe LRTI trial (LRTI and control groups) was approved by the National Health Service Research Ethics Committee in October 2013 (12/NW/0713). Recruitment opened in January 2013 and was completed in July 2013. Healthy volunteer samples were taken from the EHPC dose-ranging and reproducibility trial, approved by the same Research Ethics Committee in October 2011 (11/NW/0592). Recruitment for this study ran from October 2011 until December 2012. LRTI trial: (NCT01861184), EHPC dose-ranging and reproducibility trial: (ISRCTN85403723).

Inflammation induced by influenza virus impairs human innate immune control of pneumococcus

Colonization of the upper respiratory tract by pneumococcus is important both as a determinant of disease and for transmission into the population. The immunological mechanisms that contain pneumococcus during colonization are well studied in mice but remain unclear in humans. Loss of this control of pneumococcus following infection with influenza virus is associated with secondary bacterial pneumonia. We used a human challenge model with type 6B pneumococcus to show that acquisition of pneumococcus induced early degranulation of resident neutrophils and recruitment of monocytes to the nose. Monocyte function was associated with the clearance of pneumococcus. Prior nasal infection with live attenuated influenza virus induced inflammation, impaired innate immune function and altered genome-wide nasal gene responses to the carriage of pneumococcus. Levels of the cytokine CXCL10, promoted by viral infection, at the time pneumococcus was encountered were positively associated with bacterial load.Pneumococcal carriage in the upper respiratory tract is an important determinant of influenza severity. Jochems et al. use human systems analysis to show that influenza-induced inflammation increases bacterial burden in the nasal cavity with implications for secondary bacterial pneumonia.

Antibodies Reactive to Commensal Streptococcus mitis Show Cross-Reactivity With Virulent Streptococcus pneumoniae Serotypes

Current vaccines against Streptococcus pneumoniae, a bacterial species that afflicts people by causing a wide spectrum of diseases, do not protect against all pneumococcal serotypes. Thus, alternative vaccines to fight pneumococcal infections that target common proteins are under investigation. One promising strategy is to take advantage of immune cross-reactivity between commensal and pathogenic microbes for cross-protection. In this study, we examined the antibody-mediated cross-reactivity between S. pneumoniae and Streptococcus mitis, a commensal species closely related to S. pneumoniae. Western blot analysis showed that rabbit antisera raised against S. mitis reacted with multiple proteins of virulent S. pneumoniae strains (6B, TIGR4, and D39). Rabbit anti-S. pneumoniae IgG antibodies also showed binding to S. mitis antigens. Incubation of rabbit antisera raised against S. mitis with heterologous or homologous bacterial lysates resulted in marked inhibition of the developments of bands in the Western blots. Furthermore, plasma IgG antibodies from adult human volunteers intranasally inoculated with S. pneumoniae 6B revealed enhanced S. mitis-specific IgG titers compared with the pre-inoculation samples. Using an on-chip protein microarray representing a number of selected membrane and extracellular S. pneumoniae proteins, we identified choline-binding protein D (CbpD), cell division protein (FtsH), and manganese ABC transporter or manganese-binding adhesion lipoprotein (PsaA) as common targets of the rabbit IgG antibodies raised against S. mitis or S. pneumoniae. Cumulatively, these findings provide evidence on the antibody-mediated cross-reactivity of proteins from S. mitis and S. pneumoniae, which may have implications for development of effective and wide-range pneumococcal vaccines.