Polysaccharide-specific memory B-cells predict protection against experimental human 1 pneumococcal carriage

RATIONALE: We have previously demonstrated that experimental pneumococcal carriage enhances immunity and protects healthy adults against carriage reacquisition following re-challenge with homologous strain. Here we have used a heterologous challenge model to investigate the role of naturally acquired pneumococcal protein and polysaccharide (PS)-specific immunity in protection against carriage acquisition. METHODS: We identified healthy volunteers that were naturally colonised with pneumococcus and, following clearance of their natural carriage episode, challenged them with a heterologous 6B strain. In another cohort of volunteers we assessed 6BPS-specific, PspA-specific and PspC-specific IgG and IgA plasma and memory B-cell populations prior to and 7, 14 and 35 days following experimental pneumococcal inoculation. RESULTS: Heterologous challenge with 6B resulted in 50% carriage among volunteers with previous natural pneumococcal carriage. Protection from carriage was associated with a high number of circulating 6BPS IgG-secreting memory B-cells at baseline. There were no associations between protection from carriage and baseline levels of 6BPS IgG in serum or nasal wash, PspA-specific or PspC-specific memory B-cells or plasma cells. In volunteers who did not develop carriage, the number of circulating 6BPS memory B-cells decreased and the number of 6BPS plasma cells 7 days post inoculation. CONCLUSIONS: Our data indicate that naturally acquired polysaccharide-specific memory B-cells, but not levels of circulating IgG at time of pneumococcal exposure, are associated with protection against carriage acquisition

Susceptibility to pneumococcal colonization: identifying critical pneumococcal and mucosal markers

Introduction: Pneumococcal colonization is the pre-requisite for pneumococcal diseases such as pneumonia, otitis media and meningitis. Thus, its control is key. Pneumococcal conjugate vaccines protect against invasive pneumococcal diseases - in part through its effects on pneumococcal colonization prevalence - but present with limited serotype coverage. With the global pneumococcal burden not relenting, the need for more effective intervention strategies, as well as the possibility to test their effect on colonization is high. The experimental human pneumococcal carriage (EHPC) model – while currently limited in serotype coverage - allows the study of pneumococcal colonization control in its natural host. Project aims: Here I aimed to 1.) identify microbial characteristics that associate with experimental human pneumococcal colonization efficiency thereby establishing a tool to pre-select pneumococcal strain candidates for EHPC studies prior to human challenge; 2.) identify mucosal markers of host susceptibility to human pneumococcal colonization and 3.) determine the effect of nasal mucins on human pneumococcal colonization. Main findings: In a retrospective association study superior experimental colonization rates of pneumococcal strain SPN6B/BHN418 associated with high pneumococcal chain length in liquid culture. Implementation of the quantitative assessment of chain length as a tool for selecting pneumococcal strain candidates successfully highlighted a serotype 3 strain, which, when used for human challenge, achieved experimental colonization rates equal to those observed with SPN6B/BHN418. An integral part of the host mucosal barrier, mucins were associated with increased pneumococcal growth and survival in vitro. In vivo baseline MUC5AC levels in nasal wash positively correlated with pneumococcal load following challenge in a subset of volunteers. Nasal MUC5AC also correlated with the formation of pneumococcal aggregates in nasal fluid, highlighting the ambiguous role of mucins in human pneumococcal colonization. Implications: Pre-selection of successful pneumococcal strains for new challenge models is needed to save costs and reduce research waste, while allowing more rapid expansion of the EHPC model. Identification of factors and mechanisms influencing pneumococcal colonization of the human host further provides the basis for the improvement of current vaccines and intervention strategies against the pneumococcus

Symptoms associated with influenza vaccination and experimental human pneumococcal colonisation of the nasopharynx

Background Nasopharyngeal colonisation by S. pneumoniae is a prerequisite for invasive pneumococcal infections. Influenza co-infection leads to increased susceptibility to secondary pneumonia and mortality during influenza epidemics. Increased bacterial load and impaired immune responses to pneumococcus caused by influenza play a role in this increased susceptibility. Using an Experimental Human Challenge Model and influenza vaccines, we examined symptoms experienced by healthy adults during nasal co-infection with S. pneumoniae and live attenuated influenza virus. Methods Randomised, blinded administration of Live Attenuated Influenza Vaccine (LAIV) or Tetravalent Inactivated Influenza Vaccine (TIV) 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. Participants completed a symptom questionnaire from the first intervention until 6 days post second intervention. Results The timing and type of influenza vaccination and presence of S. pneumoniae in the nasopharynx significantly affected symptom reporting. In the study where influenza vaccination preceded bacterial inoculation: nasal symptoms were less common in the LAIV group than the TIV group (OR 0.57, p < 0.01); with colonisation status only affecting the TIV group where more symptoms were reported by colonised participants compared to non-colonised participants following inoculation (n = 12/23 [52.17%] vs n = 13/38 [34.21%], respectively; p < 0.05). In the study where influenza vaccination followed bacterial inoculation: no difference was seen in the symptoms reported between the LAIV and TIV groups following inoculation and subsequent vaccination; and symptoms were unaffected by colonisation status. Conclusion Symptoms experienced during live viral vaccination and bacterial co-infection in the nasopharynx are directly affected by the precedence of the pathogen acquisition. Symptoms were directly affected by nasal pneumococcal colonisation but only when TIV was given prior to bacterial exposure

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

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

Interaction between the nasal microbiota and S. pneumoniae in the context of live-attenuated influenza vaccine

Streptococcus pneumoniae is the main bacterial pathogen involved in pneumonia. Pneumococcal acquisition and colonization density is probably affected by viral co-infections, the local microbiome composition and mucosal immunity. Here, we report the interactions between live-attenuated influenza vaccine (LAIV), successive pneumococcal challenge, and the healthy adult nasal microbiota and mucosal immunity using an experimental human challenge model. Nasal microbiota profiles at baseline are associated with consecutive pneumococcal carriage outcome (non-carrier, low-dense and high-dense pneumococcal carriage), independent of LAIV co-administration. Corynebacterium/Dolosigranulum-dominated profiles are associated with low-density colonization. Lowest rates of natural viral co-infection at baseline and post-LAIV influenza replication are detected in the low-density carriers. Also, we detected the fewest microbiota perturbations and mucosal cytokine responses in the low-density carriers compared to non-carriers or high-density carriers. These results indicate that the complete respiratory ecosystem affects pneumococcal behaviour following challenge, with low-density carriage representing the most stable ecological state

Pneumococcal colonization impairs mucosal immune responses to live attenuated influenza vaccine.

Influenza virus infections affect millions of people annually, and current available vaccines provide varying rates of protection. However, the way in which the nasal microbiota, particularly established pneumococcal colonization, shape the response to influenza vaccination is not yet fully understood. In this study, we inoculated healthy adults with live Streptococcus pneumoniae and vaccinated them 3 days later with either tetravalent-inactivated influenza vaccine (TIV) or live attenuated influenza vaccine (LAIV). Vaccine-induced immune responses were assessed in nose, blood, and lung. Nasal pneumococcal colonization had no impact upon TIV-induced antibody responses to influenza, which manifested in all compartments. However, experimentally induced pneumococcal colonization dampened LAIV-mediated mucosal antibody responses, primarily IgA in the nose and IgG in the lung. Pulmonary influenza-specific cellular responses were more apparent in the LAIV group compared with either the TIV or an unvaccinated group. These results indicate that TIV and LAIV elicit differential immunity to adults and that LAIV immunogenicity is diminished by the nasal presence of S. pneumoniae. Therefore, nasopharyngeal pneumococcal colonization may affect LAIV efficacy

Participant perceptions and experiences of a novel community-based respiratory longitudinal sampling method in Liverpool, UK: A mixed methods feasibility study

Longitudinal, community-based sampling is important for understanding prevalence and transmission of respiratory pathogens. Using a minimally invasive sampling method, the FAMILY Micro study monitored the oral, nasal and hand microbiota of families for 6 months. Here, we explore participant experiences and opinions. A mixed methods approach was utilised. A quantitative questionnaire was completed after every sampling timepoint to report levels of discomfort and pain, as well as time taken to collect samples. Participants were also invited to discuss their experiences in a qualitative structured exit interview. We received questionnaires from 36 families. Most adults and children >5y experienced no pain (94% and 70%) and little discomfort (73% and 47% no discomfort) regardless of sample type, whereas children ≤5y experienced variable levels of pain and discomfort (48% no pain but 14% hurts even more, whole lot or worst; 38% no discomfort but 33% moderate, severe, or extreme discomfort). The time taken for saliva and hand sampling decreased over the study. We conducted interviews with 24 families. Families found the sampling method straightforward, and adults and children >5y preferred nasal sampling using a synthetic absorptive matrix over nasopharyngeal swabs. It remained challenging for families to fit sampling into their busy schedules. Adequate fridge/freezer space and regular sample pick-ups were found to be important factors for feasibility. Messaging apps proved extremely effective for engaging with participants. Our findings provide key information to inform the design of future studies, specifically that self-sampling at home using minimally invasive procedures is feasible in a family context

Nasal Pneumococcal Density is Associated with Microaspiration and Heightened Human Alveolar Macrophage Responsiveness to Bacterial Pathogens.

RATIONALE Pneumococcal pneumonia remains a global health problem. Colonization of the nasopharynx with S.pneumoniae (Spn), although, a prerequisite of infection, is the main source of exposure and immunological boosting in children and adults. However, our knowledge of how nasal colonization impacts on the lung cells, especially on the predominant alveolar macrophage (AM) population, is limited. OBJECTIVES Using a Controlled Human Infection Model to achieve nasal colonization with 6B serotype, we investigated the effect of Spn colonization on lung cells. METHODS We collected bronchoalveolar lavages from healthy pneumococcal challenged participants aged 18-49 years. Confocal microscopy, molecular and classical microbiology were used to investigate microaspiration and pneumococcal presence in the lower airways. AM opsonophagocytic capacity was assessed by functional assays in vitro, whereas flow cytometry and transcriptomic analysis were used to assess further changes on the lung cellular populations. MEASUREMENTS AND MAIN RESULTS AM from Spn-colonized exhibited increased opsonophagocytosis to pneumococcus (11.4% median increase) for four months after clearance of experimental pneumococcal colonization. AM had also increased responses against other bacterial pathogens. Pneumococcal DNA detected in the BAL samples of Spn-colonized were positively correlated with nasal pneumococcal density (r=0.71, p=0.029). Similarly, AM heightened opsonophagocytic capacity was correlated with nasopharyngeal pneumococcal density (r=0.61, p=0.025). CONCLUSIONS Our findings demonstrate that nasal colonization with pneumococcus and microaspiration prime AM, leading to brisker responsiveness to both pneumococcus and unrelated bacterial pathogens. The relative abundance of AM in the alveolar spaces, alongside with their potential for non-specific protection, render them an attractive target for novel vaccines. Clinical trial registration available at http://www.isrctn.com, ID: ISRCTN16993271

Protocol for a phase IV double-blind randomised controlled trial to investigate the effect of the 13-valent pneumococcal conjugate vaccine and the 23-valent pneumococcal polysaccharide vaccine on pneumococcal colonisation using the experimental human pneumococcal challenge model in healthy adults (PREVENTING PNEUMO 2)

Introduction: Despite widely available vaccinations, Streptococcus pneumoniae (SPN) remains a major cause of morbidity and mortality worldwide, causing community-acquired pneumonia, meningitis, otitis media, sinusitis and bacteraemia. Here, we summarise an ethically approved protocol for a double-blind, randomised controlled trial investigating the effect of the 13-valent pneumococcal conjugate vaccine (PCV13) and the 23-valent pneumococcal polysaccharide vaccine (PPV23) on pneumococcal nasopharyngeal colonisation acquisition, density and duration using experimental human pneumococcal challenge (EHPC). Methods and analysis: Healthy adult participants aged 18–50 years will be randomised to receive PCV13, PPV23 or placebo and then undergo one or two EHPCs involving intranasal administration of SPN at 1-month post-vaccination with serotype 3 (SPN3) and 6 months with serotype 6B (SPN6B). Participants randomised to PCV13 and placebo will also be randomised to one of two clinically relevant SPN3 strains from distinct lineages within clonal complex 180, clades Ia and II, creating five study groups. Following inoculation, participants will be seen on days 2, 7, 14 and 23. During the follow-up period, we will monitor safety, colonisation status, density and duration, immune responses and antigenuria. The primary outcome of the study is comparing the rate of SPN3 acquisition between the vaccinated (PCV13 or PPV23) and unvaccinated (placebo) groups as defined by classical culture. Density and duration of colonisation, comparison of acquisition rates using molecular methods and evaluation of the above measurements for individual SPN3 clades and SPN6B form the secondary objectives. Furthermore, we will explore the immune responses associated with these vaccines, their effect on colonisation and the relationship between colonisation and urinary pneumococcal antigen detection. Ethics and dissemination: The study is approved by the NHS Research and Ethics Committee (Reference: 20/NW/0097) and by the Medicines and Healthcare products Regulatory Agency (Reference: CTA 25753/0001/001–0001). Findings will be published in peer-reviewed journals

Two randomized trials of effect of live attenuated influenza vaccine on pneumococcal colonization

The human nasopharynx is frequently colonized by Streptococcus pneumoniae (the pneumococcus), serving as the reservoir for transmission, a state which necessarily precedes invasive pneumococcal infection. Influenza infection increases pneumococcal colonization density and dysregulates host immune responses, increasing the risk of secondary bacterial pneumonia and death