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RationaleStreptococcus pneumoniae serotype 3 (SPN3) is a cause of invasive pneumococcal disease and associated with low carriage rates. Following the introduction of pediatric 13-valent pneumococcal conjugate vaccine (PCV13) programmes, SPN3 declines are less than other vaccine serotypes and incidence has increased in some populations coincident with a shift in predominant circulating SPN3 clade, from I to II. A human challenge model provides an effective means for assessing the impact of PCV13 on SPN3 in the upper airway.ObjectivesTo establish SPN3's ability to colonise the nasopharynx using different inoculum clades and doses and the safety of an SPN3 challenge model.MethodsIn a human challenge study involving three well characterised and antibiotic sensitive SPN3 isolates (PFESP306 [clade Ia], PFESP231 [no clade] and PFESP505 [clade II]), inoculum doses (10,000, 20,000, 80,000, 160,000 CFU/100μL) were escalated until maximal colonisation rates were achieved, with concurrent acceptable safety.Outcome measuresPresence and density of experimental SPN3 nasopharyngeal colonisation in nasal wash samples, assessed using microbiological culture and molecular methods, on days 2, 7 and 14 post-inoculation.Results96 healthy participants (median age 21, interquartile range 19-25) were inoculated (n=6-10 per dose group, 10 groups). Colonisation rates ranged from 30.0-70.0% varying with dose and isolate. 30.0% (29/96) reported mild symptoms (82.8% sore throat, [24/29]), one developed otitis media requiring antibiotics. No serious adverse events occurred.ConclusionsAn SPN3 human challenge model is feasible and safe with comparable carriage rates to an established SPN6B human challenge model. SPN3 carriage may cause mild upper respiratory symptoms. Clinical trial registration available at www. https://www.isrctn.com/, ID: ISRCTN11306486.

Summary Background Household air pollution from solid fuels increases the risk of childhood pneumonia. Nasopharyngeal carriage of Streptococcus pneumoniae is a necessary step in the development of pneumococcal pneumonia. We aimed to assess the association between exposure to household air pollution and the prevalence and density of S pneumoniae carriage among children. Methods The Malawi Streptococcus pneumoniae Carriage and Air Pollution Exposure study was a nested, prospective, observational study of children participating in the cluster randomised controlled Cooking and Pneumonia Study (CAPS) in the Karonga Health and Demographic Surveillance System (HDSS) area in northern Malawi. CAPS compared the effects of a cleaner burning biomass-fuelled cookstove (intervention group) with traditional open-fire cooking (control group) on the incidence of pneumonia in children. Eligible children aged 6 weeks or 6 months (those recruited a 6 weeks were also followed up at age 6 months) were identified by the Karonga HDSS centre. Nasopharyngeal swabs were taken to detect S pneumoniae, and infant exposure to particulate matter with a diameter of ≤2·5 μm (PM2·5) exposure was assessed by use of a MicroPEM device. The primary outcome was the prevalence of nasopharyngeal S pneumoniae carriage in all children aged 6 months, assessed in all children with valid data on PM2·5. The effects of the intervention stoves (intention-to-treat analysis) and PM2·5 (adjusted exposure-response analysis) on the prevalence of S pneumoniae carriage were also assessed in the study children. Findings Between Nov 15, 2015, and Nov 2, 2017, 485 children were recruited (240 from the intervention group and 245 from the control group). Of all 450 children with available data at age 6 months, 387 (86% [95% CI 82–89]) were positive for S pneumoniae. Geometric mean PM2·5 exposure was 60·3 μg/m3 (95% CI 55·8–65·3) in S pneumoniae-positive children and 47·0 μg/m3 (38·3–57·7) in S pneumoniae-negative children (p=0·044). In the intention-to-treat analysis, a non-significant increase in the risk of S pneumoniae carriage was observed in intervention group children compared with control group children (odds ratio 1·36 [95% CI 0·95–1·94]; p=0·093). In the exposure-response analysis, a significant association between PM2·5 exposure and S pneumoniae carriage was observed; a one unit increase in decile of PM2·5 was found to significantly increase the risk of S pneumoniae carriage by 10% (1·10 [1·01–1·20]; p=0·035), after adjustment for age, sex, 13-valent pneumococcal conjugate vaccination status, season, current use of antibiotics, and MicroPEM run-time. Interpretation Despite the absence of effect from the intervention cookstove, household air pollution exposure was significantly associated with the prevalence of nasopharyngeal S pneumoniae carriage. These results provide empirical evidence for the potential mechanistic association between exposure to household air pollution and childhood pneumonia. Funding Bill & Melinda Gates Foundation.

RationalePneumococcal colonisation is key to the pathogenesis of invasive disease, but is also immunogenic in young adults, protecting against re-colonisation. Colonisation is rarely detected in older adults, despite high rates of pneumococcal disease.ObjectivesTo establish experimental human pneumococcal colonisation in healthy adults aged 50—84 years, to measure the immune response to pneumococcal challenge, and to assess the protective effect of prior colonisation against autologous strain rechallenge.MethodsSixty-four participants were inoculated with Streptococcus pneumoniae (serotype 6B, 80,000CFU in each nostril). Colonisation was determined by bacterial culture of nasal wash, serum anti-6B capsular IgG responses by ELISA, and anti-protein immune responses by multiplex electrochemiluminescence.Measurements and Main ResultsExperimental colonisation was established in 39% of participants (25/64) with no adverse events. Colonisation occurred in 47% (9/19) of participants aged 50—59 compared with 21% (3/14) in those aged ≥70 years. Previous pneumococcal polysaccharide vaccination did not protect against colonisation. Colonisation did not confer serotype-specific immune boosting: GMT (95% CI) 2.7μg/mL (1.9—3.8) pre-challenge versus 3.0 (1.9—4.7) four weeks post-colonisation (p = 0.53). Furthermore, pneumococcal challenge without colonisation led to a drop in specific antibody levels from 2.8μg/mL (2.0—3.9) to 2.2μg/mL (1.6—3.0) post-challenge (p = 0.006). Anti-protein antibody levels increased following successful colonisation. Rechallenge with the same strain after a median of 8.5 months (IQR 6.7—10.1) led to recolonisation in 5/16 (31%).ConclusionsIn older adults, experimental pneumococcal colonisation is feasible and safe, but demonstrates different immunological outcomes compared with younger adults in previous studies.

Streptococcus pneumoniae has a complex relationship with its obligate human host. On the one hand, the pneumococci are highly adapted commensals, and their main reservoir on the mucosal surface of the upper airways of carriers enables transmission. On the other hand, they can cause severe disease when bacterial and host factors allow them to invade essentially sterile sites, such as the middle ear spaces, lungs, bloodstream and meninges. Transmission, colonization and invasion depend on the remarkable ability of S. pneumoniae to evade or take advantage of the host inflammatory and immune responses. The different stages of pneumococcal carriage and disease have been investigated in detail in animal models and, more recently, in experimental human infection. Furthermore, widespread vaccination and the resulting immune pressure have shed light on pneumococcal population dynamics and pathogenesis. Here, we review the mechanistic insights provided by these studies on the multiple and varied interactions of the pneumococcus and its host.

IntroductionAfrica harbours a high burden of pneumococcal disease, with associated high mortality rates. Despite 34 countries introducing the pneumococcal conjugate vaccine, which reduces the risk of pneumococcal carriage (a prerequisite for disease) of some of the most pathogenic pneumococcal serotypes, it remains uncertain whether they will achieve the sustained direct or indirect protection necessary to reduce pneumococcal carriage to levels sufficient to interrupt transmission and disease. We will therefore summarise the available data on the impact of the pneumococcal conjugate vaccine in reducing vaccine serotype carriage and pneumococcal pneumonia in Africa between 2000 and 2019.Methods and analysisUsing a predetermined search strategy, we will conduct a comprehensive search of PubMed, MEDLINE database, the Excerpta Medica Database, the ISI Web of Science (Science Citation Index), Scopus and the African Index Medicus to identify published studies reporting the prevalence of Streptococcus pneumoniae carriage (vaccine type and non-vaccine type), incidence rates of pneumococcal pneumonia and mortality among children, adults and HIV-infected (all-ages) pre-pneumococcal conjugate vaccine (PCV) and post-PCV introduction (published between 1st January 2000 and 31st December 2019) in African countries that have introduced PCVs (PCV7/PCV10/PCV13) in their routine national immunisation programme. The studies retained and data extracted will be assessed for bias using prevalidated tools and checklists. Heterogeneity across studies will be assessed using the χ2 test on Cochrane Q statistic. A random effect meta-analysis will be used to estimate the overall prevalence of pneumococcal carriage and incidence of pneumococcal pneumonia across studies with similar characteristics. Results will be reported in compliance with the Meta-Analysis Of Observational Studies in Epidemiology guidelines. The protocol has been prepared in accordance to the 2015 guidelines on Preferred Reporting Items for Systematic Reviews and Meta-Analyses.Ethics and disseminationThis systematic review will not require ethical approval as we will be using already published data. The final manuscript will be submitted for publication in a peer-reviewed journal and presented at conferences.PROSPERO registration numberCRD42019130976.

Introduction: Lower respiratory tract infections are the fourth cause of death worldwide and pneumococcus is the leading cause of pneumonia. Nonetheless, existing pneumococcal vaccines are less effective against pneumonia than invasive diseases and serotype replacement is a major concern. Protein antigens could induce serotype-independent protection, and mucosal immunization could offer local and systemic immune responses and induce protection against pneumococcal colonization and lung infection. Areas covered: Immunity induced in the experimental human pneumococcal carriage model, approaches to address the physiological barriers to mucosal immunization and improve delivery of the vaccine antigens, different strategies already tested for pneumococcal mucosal vaccination, including live recombinant bacteria, nanoparticles, bacterium-like particles, and nanogels as well as, nasal, pulmonary, sublingual and oral routes of vaccination. Expert opinion: The most promising delivery systems are based on nanoparticles, bacterial-like particles or nanogels, which possess greater immunogenicity than the antigen alone and are considered safer than approaches based on living cells or toxoids. These particles can protect the antigen from degradation, eliminating the refrigeration need during storage and allowing the manufacture of dry powder formulations. They can also increase antigen uptake, control release of antigen and trigger innate immune responses.

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.

Background S. pneumoniae (SP) is one of the most important bacteria for pneumonia among adults. We investigated hospital-based proportion of antimicrobial resistance, distribution of serotypes, sequence types (STs) and clinical findings among adult pneumococcal pneumonia patients, and compared microbiological results with the previous study that was reported 10 years ago in Japan. Methods A multicenter prospective surveillance for adult pneumonia was conducted from September 2011 to August 2014 in Japan. We enrolled aged over 15 years, community-acquired or healthcare-associated pneumonia patients, and obtained clinical information and sputum samples. Sputum samples were cultured quantitatively or qualitatively at each study sites. Identified SP strains were transported to our laboratory for serotyping by the Quellung reaction. We also extracted DNA from SP strains for multilocus sequence typing. Antimicrobial sensitivity tests for penicillin (PCG), ceftriaxone (CTRX), and meropenem (MEPM) were conducted using agar dilution method. Differences of clinical findings were analyzed using the chi-square test. Results We enrolled 200 cases and obtained 205 SP strains. Most dominant serotype was three (24.4%) and ST was 180 (22.9%). Those results were same as the previous report. The proportion of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine serotypes was 58.5 and 75.6%, respectively. Most of SP strains were sensitive for antibiotics (96.1% for PCG, 94.1% for CTRX, and 81.5% for MEPM) and the antimicrobial sensitivity also did not greatly have a change with the previous study reported 10 years ago. Pneumococcal pneumonia patients due to serotype 3 was higher hospitalization rate and CURB-65 score than other serotypes (hospitalization rate was 73.5 and 60.3%, respectively; P = 0.095, and proportion of 3 or more CURB-65 score was 36.4 and 20.7%, respectively; P = 0.039). Conclusion In Japan, SP sensitivity for antibiotics, dominant serotype and ST were not changed so much from 10 years ago. Serotype 3 SP contributed to the disease severity of adult pneumococcal pneumonia in Japan. Disclosures K. Morimoto, Pfizer: speaker, Speaker honorarium.

Background:\ud \ud Thirteen-valent pneumococcal conjugate vaccine (PCV13) was introduced in Malawi in November 2011 and is offered to infants at 6, 10 and 14 weeks of age as part of routine immunisation. PCV13 is expected to reduce vaccine type (VT) nasopharyngeal carriage, leading to reduced transmission and herd protection.\ud Methods:\ud \ud We compared pneumococcal carriage in rural Karonga District, Malawi, pre-vaccine in 2009–2011 and post-vaccine in 2014 using a combination of cross-sectional and longitudinal analyses. Nasopharyngeal swabs were collected from a cohort of mother-infant pairs and household members

Colonization of the human nasopharynx by pneumococcus is extremely common and is both the primary reservoir for transmission and a prerequisite for disease. Current vaccines targeting the polysaccharide capsule effectively prevent colonization, conferring herd protection within vaccinated communities. However, these vaccines cover only a subset of all circulating pneumococcal strains and serotype replacement has been observed. Given the success of PCV in preventing colonization in unvaccinated adults within vaccinated communities, reducing nasopharyngeal colonization has become an outcome of interest for novel vaccines. Here, we discuss the immunological mechanisms that control nasopharyngeal colonization with an emphasis on findings from human studies. Increased understanding of these immunological mechanisms is required to identify correlates of protection against colonization which will facilitate the early testing and design of novel vaccines.

Rationale:\ud Nasopharyngeal carriage of Streptococcus pneumoniae is a prerequisite for invasive disease, but the majority of carriage episodes are asymptomatic and self-resolving. Interactions determining the development of carriage versus invasive disease are poorly understood but will influence the effectiveness of vaccines or therapeutics that disrupt nasal colonization.\ud \ud Objectives:\ud We sought to elucidate immunological mechanisms underlying noninvasive pneumococcal nasopharyngeal carriage.\ud \ud Methods:\ud Pneumococcal interactions with human nasopharyngeal and bronchial fibroblasts and epithelial cells were investigated in vitro. A murine model of nasopharyngeal carriage and an experimental human pneumococcal challenge model were used to characterize immune responses in the airways during carriage.\ud Measurements and Main Results: We describe the previously unknown immunological basis of noninvasive carriage and highlight mechanisms whose perturbation may lead to invasive disease. We identify the induction of active transforming growth factor (TGF)-β1 by S. pneumoniae in human host cells and highlight the key role for TGF-β1 and T regulatory cells in the establishment and maintenance of nasopharyngeal carriage in mice and humans. We identify the ability of pneumococci to drive TGF-β1 production from nasopharyngeal cells in vivo and show that an immune tolerance profile, characterized by elevated TGF-β1 and high nasopharyngeal T regulatory cell numbers, is crucial for prolonged carriage of pneumococci. Blockade of TGF-β1 signaling prevents prolonged carriage and leads to clearance of pneumococci from the nasopharynx.\ud \ud Conclusions:\ud These data explain the mechanisms by which S. pneumoniae colonize the human nasopharynx without inducing damaging host inflammation and provide insight into the role of bacterial and host constituents that allow and maintain carriage.

Pneumonia remains the leading cause of childhood mortality and the most common reason for adult hospitalisation in low and middle income countries, despite advances in preventative and management strategies. In the last decade, pneumonia mortality in children has fallen to approximately 1.3 million cases in 2011, with most deaths occurring in low income countries. Important recent advances include more widespread implementation of protein-polysaccharide conjugate vaccines against Haemophilus influenzae type B and Streptococcus pneumoniae, implementation of case-management algorithms and better prevention and treatment of HIV. Determining the aetiology of pneumonia is challenging in the absence of reliable diagnostic tests. High uptake of new bacterial conjugate vaccines may impact on pneumonia burden, aetiology and empiric therapy but implementation in immunisation programmes in many low and middle income countries remains an obstacle. Widespread implementation of currently effective preventative and management strategies for pneumonia remains challenging in many low and middle income countries.

Streptococcus pneumoniae is a common cause of infection in both HIV positive patients and those with complement deficiencies. We hypothesised that HIV positive individuals might exhibit reduced opsonisation of pneumococcus with complement due to reduced levels of S. pneumoniae specific IgG. We discovered no difference in C3 deposition on S. pneumoniae between HIV positive or negative individuals, and furthermore C3 deposition remained unchanged as HIV progressed towards AIDS. We found no correlation between C3 deposition on S. pneumoniae and CD4 cell count in HIV infected individuals. Hence we have demonstrated no failure of complement immunity in HIV positive patients.

Background Blood culture surveillance will be used for assessing the public health effectiveness of pneumococcal conjugate vaccines in Africa. Between 2003 and 2006 we assessed blood culture outcome and performance in adult patients in the central public hospital in Blantyre, Malawi, before and after the introduction of a dedicated nurse led blood culture team. Methods A prospective observational study. Results Following the introduction of a specialised blood culture team in 2005, the proportion of contaminated cultures decreased (19.6% in 2003 to 5.0% in 2006), blood volume cultured increased and pneumococcal recovery increased significantly from 2.8% of all blood cultures to 6.1%. With each extra 1 ml of blood cultured the odds of recovering a pneumococcus increased by 18%. Conclusion Standardisation and assessment of blood culture performance (blood volume and contamination rate) should be incorporated into pneumococcal disease surveillance activities where routine blood culture practice is constrained by limited resources.

Rationale\ud Pneumococcal pneumonia is a leading cause of death globally, and susceptibility to invasive and pulmonary infection is increased in chronic respiratory conditions. Pneumococcal colonisation of the nasopharynx necessarily precedes disease, but the relationship of colonisation with Streptococcus pneumoniae and chronic respiratory disease (such as asthma) is unknown. Asthma is a heterogenous condition representing several phenotypes which have corresponding clinical characteristics and responses to therapy. Immune responses mediated by cytokines from cells such T helper cells 2 (Th2) and Th17 lead to airway inflammation. Both this inflammatory pathology, and the treatment of it (with inhaled corticosteroids) potentially affect nasopharyngeal colonisation with S.pneumoniae. Hypothetically, airway inflammation might increase the potential for bacterial attachment, or increase bacterial clearance, or both. As nasopharyngeal colonisation is immunogenic in healthy adults, the balance of these opposing mechanisms may impact the resulting immune response. We have safely used the experimental human pneumococcal challenge (EHPC) model in healthy individuals to study nasopharyngeal colonisation and its associated mucosal and systemic immune responses.\ud \ud Objectives\ud Using experimental pneumococcal challenge in people with asthma, I examined the acquisition of nasopharyngeal colonisation of Streptococcus pneumoniae, its association with clinical characteristics, and systemic immune responses. I compared the results with historic data from earlier EHPC studies of healthy controls.\ud \ud Methods\ud I enrolled people with physician-diagnosed, well-controlled asthma on maintenance inhaled corticosteroids. Participants were challenged with pneumococcus serotype 6B, as this is not isolated from nasal samples within the local community. Blood and mucosal samples were collected at baseline and on days 2,7, 9, 14, 22 and 29 after challenge. The results were compared to healthy controls from 4 experimental human challenge studies, with 2 performed concurrently and 2 in previous years.\ud \ud Main Results\ud Experimental colonisation rates were not significantly increased in people with asthma compared to healthy controls. The number of colonised participants (nasal wash positive for bacterial culture at any time point) was 28 (56%) in asthma vs 68 (45%) in healthy controls (Pearson’s chi square p=0.178). The density calculated using the area under time cure (AUC) was similar in people with asthma compared to healthy controls (median [IQR] asthma 63.49 [14.04-116.3] vs healthy controls 81.18 [48.15-104.5] Mann Whitney U test p=0.060). Acquisition of colonisation was independent of baseline characteristics such as blood eosinophils and fractional exhaled nitric oxide levels.\ud The duration of experimental colonisation was significantly shorter in asthma compared to healthy controls (median [IQR] 14 [7-29] vs 29[14-29]) p=0.034 Mann Whitney U test. Colonisation led to an increase in IgG titres to capsular polysaccharide and pneumococcal proteins in people with asthma as previously described in healthy controls. Body mass index correlated positively with likelihood of colonisation in people with asthma. Median BMI for the whole cohort was 24.6 (IQR) (21.6-27.5), with colonised participants (nasal wash positive for bacterial culture at any time point) having a higher BMI median 24.7 (24.1-29.0) vs 23.5 (20.1-26.4) in non-colonised (p=0.019 independent samples t test).\ud \ud Conclusions:\ud Experimental colonisation is not affected by clinical characteristics of asthma, and it is positively correlated with a high BMI. The rate and density of experimental nasopharyngeal colonisation in people with asthma are similar as seen in healthy controls. The duration of colonisation is significantly reduced in people with asthma compared to healthy controls, with a similar immunogenic effect as seen in healthy controls. Interventions to reduce the likelihood of severe and invasive pneumococcal disease, to which people with asthma are more prone, could be targeted at specific sub-groups. Further investigation could suggest if those with higher BMI should have a lower threshold for vaccination.

The burden of pneumococcal pneumonia remains high worldwide. The disease disproportionately affects infants, the elderly and the immunocompromised. Nasopharyngeal colonisation with S. pneumoniae is the pre-requisite for disease but also, paradoxically, an immunising event. Current pneumococcal vaccines offer limited protection against pneumonia and do not offer serotype-independent coverage. Serotype replacement is particularly concerning. Identification of serotype-independent correlates of protection against S. pneumoniae infection will accelerate development of vaccines with broad coverage. Protein vaccines are a particularly promising way to confer serotype-independent protection.\ud The experimental human pneumococcal challenge (EHPC) model enables us to investigate correlates of protection against experimental colonisation with S. pneumoniae and study protein-directed immune responses to nasopharyngeal colonisation with the bacteria. A multiplex Luminex assay was developed and used to measure serum IgG against 75 conserved pneumococcal antigens pre- and post-intranasal inoculation with two serotypes of pneumococcus (6B and 15B) in EHPC volunteers. Peripheral blood mononuclear cells taken pre- and post-challenge with serotype 6B pneumococcus were used to evaluate correlation between protein-specific IgG+ memory B-cell responses and protection against experimental colonisation as well as memory B-cell responses to pneumococcal challenge by ELISpot. Memory B-cell responses to six selected proteins and the novel PnuBioVax vaccine were investigated. The results of this work showed that colonisation with serotype 6B pneumococcus induces significant systemic protein-mediated antibody and memory B-cell responses. No single protein antigen correlated with protection against experimental colonisation with S. pneumoniae. However, whilst not significant, there was a trend that PnuBioVax-specific memory B-cells conferred protection against experimental pneumococcal colonisation. Five novel nanoparticle-based vaccines were also investigated to determine their effects on dendritic cell maturation and support of T-cell activation. One of these nanoparticles was subsequently loaded with pneumococcal protein PspA to investigate antigen-specific response. Further work is needed to ascertain the immune response to these novel vaccines, though they are a promising method of serotype-independent vaccine delivery.\ud It is likely that for a novel pneumococcal protein vaccine to confer serotype-independent protection against pneumococcal pneumonia, a combination of protein antigens will need to be included in vaccine formulations. Mucosal vaccination in particular will likely be needed to induce robust immunity