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BACKGROUND\ud \ud Although recent epidemiological data suggest that pneumococci may contribute to the risk of SARS-CoV-2 disease, cases of co-infection with Streptococcus pneumoniae in COVID-19 patients during hospitalisation have been reported infrequently. This apparent contradiction may be explained by interactions of SARS-CoV-2 and pneumococcus in the upper airway, resulting in the escape of SARS-CoV-2 from protective host immune responses.\ud \ud METHODS\ud \ud Here, we investigated the relationship of these two respiratory pathogens in two distinct cohorts of a) healthcare workers with asymptomatic or mildly symptomatic SARS-CoV-2 infection identified by systematic screening and b) patients with moderate to severe disease who presented to hospital. We assessed the effect of co-infection on host antibody, cellular and inflammatory responses to the virus.\ud \ud RESULTS\ud \ud In both cohorts, pneumococcal colonisation was associated with diminished anti-viral immune responses, which affected primarily mucosal IgA levels among individuals with mild or asymptomatic infection and cellular memory responses in infected patients.\ud \ud CONCLUSION\ud \ud Our findings suggest that S. pneumoniae impairs host immunity to SARS-CoV-2 and raises the question if pneumococcal carriage also enables immune escape of other respiratory viruses and facilitates reinfection occurrence.\ud \ud TRIALS REGISTRATION\ud \ud ISRCTN89159899 for FASTER study and Clinicaltrials.gov identifier: NCT03502291 for LAIV study.

Rationale: Nasopharyngeal administration of live virulence-attenuated Streptococcus pneumoniae strains is a potential novel preventative strategy. One target for creating reduced virulence S. pneumoniae strains is the capsule, but loss of the capsule reduces the duration of S. pneumoniae colonisation in mice which could impair protective efficacy against subsequent infection. Objectives: To assess protective efficacy of nasopharyngeal administration of unencapsulated S. pneumoniae strains in murine infection models. Methods: Strains containing cps locus deletions combined with the S. pneumoniae virulence factors psaA (reduces colonisation) or proABC (no effect on colonisation) were constructed and their virulence phenotypes and ability to prevent recolonisation or invasive infection assessed using mouse infection models. Serological responses to colonisation were compared between strains using ELISAs, immunoblots and 254 S. pneumoniae protein antigen array. Measurements and Main Results: The ∆cps/piaA and ∆cps/proABC strains were strongly attenuated in virulence in both invasive infection models and had a reduced ability to colonise the nasopharynx. ELISAs, immunoblots and protein arrays showed colonisation with either strain stimulated weaker serological responses than the wild type strain. Mice previously colonised with these strains were protected against septicaemic pneumonia but, unlike mice colonised with the wild type strain, not against S. pneumoniae recolonisation. Conclusions: Colonisation with the ∆cps/piaA and ∆cps/proABC strains prevented subsequent septicaemia, but in contrast, to published data for encapsulated double mutant strains they did not prevent recolonisation with S. pneumoniae. These data suggest targeting the cps locus is a less effective option for creating live attenuated strains that prevent S. pneumoniae infections.

Nasopharyngeal colonization of potential respiratory pathogens such as Streptococcus pneumoniae is the major source of transmission and precursor of invasive disease. Swabbing deeply the nasopharynx, which is currently recommended by World Health Organization, provides accurate pneumococcal detection but is unpleasant. We showed that nasal lining fluid filter strips offer equal detection sensitivity.

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., Using a Streptococcus pneumoniae human challenge model, the authors here show that baseline nasal microbiota profiles are associated with pneumococcal acquisition and density, independent of live-attenuated influenza vaccine (LAIV) administration, and with mucosal cytokine responses to LAIV.

Pneumococcal colonization is rarely studied in adults, except as part of family surveys. We report the outcomes of colonization screening in healthy adults (non-smokers without major comorbidities or contact with children under five years) who had volunteered to take part in clinical research. Using nasal wash culture, we detected colonization in 6.5% (52/795) of volunteers. Serotype 3 was the commonest serotype (10/52). The majority of the remainder (35/52) were non-vaccine serotypes, but we also identified persistent circulation of serotypes 19A and 19F. Resistance to at least one of six antibiotics tested was found in 8/52 isolates.

BACKGROUND\ud Group B Streptococcus (GBS) causes invasive infections in neonates and has been implicated as a cause of prelabour rupture of membranes, preterm delivery and stillbirths. The success of phase II trials of polyvalent polysaccharide GBS vaccines indicates that these infections are potentially preventable. Nigeria is the most populous country in Africa with one of the highest birth rates, one of the highest neonatal sepsis incidence rates and one of the highest mortality rates in the world. Therefore, before the possible introduction of preventive strategies such as intrapartum antibiotic prophylaxis or GBS vaccine into Nigeria, it is vital that there is accurate data on the aetiology of neonatal sepsis and on the incidence of GBS neonatal sepsis in particular. The objective of this study was to determine the incidence and aetiology of neonatal sepsis in Nigeria with a focus on GBS sepsis and also to assess the potential impact of a GBS vaccine.\ud METHODS\ud A literature search was conducted on the databases of African journals online, PubMed and Google Scholar for works conducted between 1987 to 2017. Case reports, reviews, and studies not stating specific culture methods or specific bacteria isolated were excluded. Data extracted included; incidence of neonatal sepsis, method of blood culture, blood volume, sample size, bacterial agents isolated and history of antibiotic use. PRISMA guidelines were followed and modified Down's and Black criteria used to evaluate the quality of studies.\ud RESULTS\ud A total of 5,114 studies were reviewed for neonatal sepsis out of which 24 consisting of a total of 2,280 cases were selected for final review. Nine studies met criteria for assessment of hospital based incidence of neonatal sepsis representing 31,305 hospital births. The incidence of neonatal sepsis was 18.2/1000 livebirths with range from 7-55/1000 livebirths while the GBS incidence was 0.06/1000 livebirths with range from 0-2/1000 live births. We discovered various limitations such as identification techniques that could result in underestimation of the true incidence of GBS sepsis. Pathogens such as Klebsiella pneumoniae and Staphylococcus aureus were more commonly isolated than GBS.\ud IMPLICATIONS OF KEY FINDINGS\ud The hospital based incidence of neonatal sepsis was high at 18.2/1000 live births while that due to GBS was 0.06/1000 live births. The burden of neonatal sepsis, including that attributable to GBS is substantial and could be reduced by preventive strategies such as intrapartum antibiotic prophylaxis or GBS vaccine. There is however very sparse meaningful data currently. Well planned prospective studies with larger sample sizes, more advanced isolation and identification techniques and those following up invasive disease cases for possible short and long term sequelae are needed-not only prior to possible introduction of the vaccine to determine the baseline epidemiology, but also thereafter to monitor its impact on the population. Strategies need to be developed to also reduce the morbidity and mortality attributable to other bacteria that have an incidence even greater than that of GBS.

Highlights • Pneumococcal-specific Th17 responses in HIV-infected adults are preserved. • The frequency of pneumococcal-specific Th17 cells is increased in ART-treated HIV-infected adults. • Depletion of pneumococcal-specific Th17 cells is unlikely the reason behind the increased susceptibility to pneumonia in HIV-infected adults., Summary Objective We explored if HIV infection is associated with impaired T-Helper 17 responses against Streptococcus pneumoniae in the lung. Methods We recruited 30 HIV-uninfected healthy controls, 23 asymptomatic HIV-infected adults not on ART, and 40 asymptomatic HIV-infected adults on ART (Median time 3.5yrs), in whom we collected bronchoalveolar lavage fluid. We measured alveolar CD4+ T cell immune responses following stimulation with pneumococcal cell culture supernatant using flow cytometry-based intracellular cytokine staining. Results We found that the proportion of alveolar CD4+ T cells producing IL-17A following stimulation with pneumococcal cell culture supernatant (CCS) was similar between HIV-uninfected controls and ART-naïve HIV-infected adults (0.10% vs. 0.14%; p = 0.9273). In contrast, the proportion and relative absolute counts of CD4+ T cells producing IL-17A in response to pneumococcal CCS were higher in ART-treated HIV-infected adults compared HIV-uninfected controls (0.22% vs. 0.10%, p = 0.0166; 5420 vs. 1902 cells/100 ml BAL fluid; p = 0.0519). The increase in relative absolute numbers of IL-17A-producing alveolar CD4+ T cells in ART-treated individuals was not correlated with the peripheral blood CD4+ T cell count (r=–0.1876, p = 0.1785). Conclusion Alveolar Th17 responses against S. pneumoniae are preserved in HIV-infected adults. This suggests that there are other alternative mechanisms that are altered in HIV-infected individuals that render them more susceptible to pneumococcal pneumonia.

Streptococcus pneumoniae is responsible for diseases causing major global public health problems, including meningitis, pneumonia and septicaemia. Despite recent advances in antimicrobial therapy, pneumococcal meningitis remains a life-threatening disease. Furthermore, long-term sequelae are a major concern for survivors. Hence, a better understanding of the processes occurring in the central nervous system is crucial to the development of more effective management strategies. We used mass spectrometry based quantitative proteomics to identify protein changes in cerebrospinal fluid from children with Streptococcus pneumoniae infection, compared with children admitted to hospital with bacterial meningitis symptoms but negative diagnosis. Samples were analysed, by label free proteomics, in two independent cohorts (cohort 1: cases (n = 8) and hospital controls (n = 4); cohort 2: cases (n = 8), hospital controls (n = 8)). Over 200 human proteins were differentially expressed in each cohort, of which 65% were common to both. Proteins involved in the immune response and exosome signalling were significantly enriched in the infected samples. For a subset of proteins derived from the proteome analysis, we corroborated the proteomics data in a third cohort (hospital controls (n = 15), healthy controls (n = 5), cases (n = 20)) by automated quantitative western blotting, with excellent agreement with our proteomics findings. Proteomics data are available via ProteomeXchange with identifier PXD004219.

Objectives \ud \ud This study aimed to define the molecular basis of co-trimoxazole resistance in Malawian pneumococci under the dual selective pressure of widespread co-trimoxazole and sulfadoxine/pyrimethamine use.\ud \ud Methods \ud \ud We measured the trimethoprim and sulfamethoxazole MICs and analysed folA and folP nucleotide and translated amino acid sequences for 143 pneumococci isolated from carriage and invasive disease in Malawi (2002–08).\ud \ud Results \ud \ud Pneumococci were highly resistant to both trimethoprim and sulfamethoxazole (96%, 137/143). Sulfamethoxazole-resistant isolates showed a 3 or 6 bp insertion in the sulphonamide-binding site of folP. The trimethoprim-resistant isolates fell into three genotypic groups based on dihydrofolate reductase (encoded by folA) mutations: Ile-100-Leu (10%), the Ile-100-Leu substitution together with a residue 92 substitution (56%) and those with a novel uncharacterized resistance genotype (34%). The nucleotide sequence divergence and dN/dS of folA and folP remained stable from 2004 onwards.\ud \ud Conclusions \ud \ud S. pneumoniae exhibit almost universal co-trimoxazole resistance in vitro and in silico that we believe is driven by extensive co-trimoxazole and sulfadoxine/pyrimethamine use. More than one-third of pneumococci employ a novel mechanism of co-trimoxazole resistance. Resistance has now reached a point of stabilizing evolution. The use of co-trimoxazole to prevent pneumococcal infection in HIV/AIDS patients in sub-Saharan Africa should be re-evaluated.

Contains fulltext : 138235.pdf (Publisher’s version ) (Open Access) Colonization of the nasopharynx by Streptococcus pneumoniae is a necessary precursor to pneumococcal diseases that result in morbidity and mortality worldwide. The nasopharynx is also host to other bacterial species, including the common pathogens Staphylococcus aureus, Haemophilus influenzae, and Moraxella catarrhalis. To better understand how these bacteria change in relation to pneumococcal colonization, we used species-specific quantitative PCR to examine bacterial densities in 52 subjects 7 days before, and 2, 7, and 14 days after controlled inoculation of healthy human adults with S. pneumoniae serotype 6B. Overall, 33 (63%) of subjects carried S. pneumoniae post-inoculation. The baseline presence and density of S. aureus, H. influenzae, and M. catarrhalis were not statistically associated with likelihood of successful pneumococcal colonization at this study's sample size, although a lower rate of pneumococcal colonization in the presence of S. aureus (7/14) was seen compared to that in the presence of H. influenzae (12/16). Among subjects colonized with pneumococci, the number also carrying either H. influenzae or S. aureus fell during the study and at 14 days post-inoculation, the proportion carrying S. aureus was significantly lower among those who were colonized with S. pneumoniae (p = 0.008) compared to non-colonized subjects. These data on bacterial associations are the first to be reported surrounding experimental human pneumococcal colonization and show that co-colonizing effects are likely subtle rather than absolute.

The pneumococcal pilus has been shown to be an important determinant of adhesion and virulence in mouse models of colonization, pneumonia, and bacteremia. A pilus is capable of inducing protective immunity, supporting its inclusion in next-generation pneumococcal protein vaccine formulations. Whether this vaccine target is common among pneumococci in sub-Saharan Africa is uncertain. To define the prevalence and genetic diversity of type I and II pili among invasive pneumococci in Malawi prior to the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) into routine childhood immunization, we examined 188 Streptococcus pneumoniae isolates collected between 2002 and 2008 (17% serotype 1). In this region of high disease burden, we found a low frequency of invasive piliated pneumococci (14%) and pilus gene sequence diversity similar to that seen previously in multiple global pneumococcal lineages. All common serotypes with pilus were covered by PCV13 and so we predict that pilus prevalence will be reduced in the Malawian pneumococcal population after PCV13 introduction.

Infectious challenge of the human nasal mucosa elicits immune responses that determine the fate of the host-bacterial interaction; leading either to clearance, colonisation and/or disease. Persistent antigenic exposure from pneumococcal colonisation can induce both humoral and cellular defences that are protective against carriage and disease. We challenged healthy adults intra-nasally with live 23F or 6B Streptococcus pneumoniae in two sequential cohorts and collected nasal wash, bronchoalveolar lavage (BAL) and blood before and 6 weeks after challenge. We hypothesised that both cohorts would successfully become colonised but this did not occur except for one volunteer. The effect of bacterial challenge without colonisation in healthy adults has not been previously assessed. We measured the antigen-specific humoral and cellular immune responses in challenged but not colonised volunteers by ELISA and Flow Cytometry. Antigen-specific responses were seen in each compartment both before and after bacterial challenge for both cohorts. Antigen-specific IgG and IgA levels were significantly elevated in nasal wash 6 weeks after challenge compared to baseline. Immunoglobulin responses to pneumococci were directed towards various protein targets but not capsular polysaccharide. 23F but not 6B challenge elevated IgG anti-PspA in BAL. Serum immunoglobulins did not increase in response to challenge. In neither challenge cohort was there any alteration in the frequencies of TNF, IL-17 or IFNγ producing CD4 T cells before or after challenge in BAL or blood. We show that simple, low dose mucosal exposure with pneumococci may immunise mucosal surfaces by augmenting anti-protein immunoglobulin responses; but not capsular or cellular responses. We hypothesise that mucosal exposure alone may not replicate the systemic immunising effect of experimental or natural carriage in humans., Author Summary Exposure to respiratory pathogens such as Streptococcus pneumoniae (pneumococcus) is a frequent event that can result in immediate clearance, nasal colonisation or disease for the host. Human and mouse studies have shown that natural colonisation is an immunising event. Colonisation is prevalent in children but rare in human adults (

The polysaccharide capsule of Streptococcus pneumoniae defines over ninety serotypes, which differ in their carriage prevalence and invasiveness for poorly understood reasons. Recently, an inverse correlation between carriage prevalence and oligosaccharide structure of a given capsule has been described. Our previous work suggested a link between serotype and growth in vitro. Here we investigate whether capsule production interferes with growth in vitro and whether this predicts carriage prevalence in vivo. Eighty-one capsule switch mutants were constructed representing nine different serotypes, five of low (4, 7F, 14, 15, 18C) and four of high carriage prevalence (6B, 9V, 19F, 23F). Growth (length of lag phase, maximum optical density) of wildtype strains, nontypeable mutants and capsule switch mutants was studied in nutrient-restricted Lacks medium (MLM) and in rich undefined brain heart infusion broth supplemented with 5% foetal calf serum (BHI+FCS). In MLM growth phenotype depended on, and was transferred with, capsule operon type. Colonization efficiency of mouse nasopharynx also depended on, and was transferred with, capsule operon type. Capsule production interfered with growth, which correlated inversely with serotype-specific carriage prevalence. Serotypes with better growth and higher carriage prevalence produced thicker capsules (by electron microscopy, FITC-dextran exclusion assays and HPLC) than serotypes with delayed growth and low carriage prevalence. However, expression of cpsA, the first capsule gene, (by quantitative RT-PCR) correlated inversely with capsule thickness. Energy spent for capsule production (incorporation of H3-glucose) relative to amount of capsule produced was higher for serotypes with low carriage prevalence. Experiments in BHI+FCS showed overall better bacterial growth and more capsule production than growth in MLM and differences between serotypes were no longer apparent. Production of polysaccharide capsule in S. pneumoniae interferes with growth in nutrient-limiting conditions probably by competition for energy against the central metabolism. Serotype-specific nasopharyngeal carriage prevalence in vivo is predicted by the growth phenotype., Author Summary Streptococcus pneumoniae bacteria are responsible for serious human infections including meningitis, pneumonia and bacteraemia and are a common cause of otitis media (ear infection) in children. However, they most often reside harmlessly in the infant nasopharynx. An association has long been observed between the type of polysaccharide capsule surrounding the bacteria and harmless colonization versus invasive disease. Here we suggest that capsule types that are costly for the bacteria to make are produced in lower quantities and their production limits the growth of the bacteria in nutrient-restricted conditions. In contrast, bacteria with capsules that require less energy can produce more capsule and grow more successfully. This may be an explanation for why S. pneumoniae with certain capsule types can be effective long-term colonizers of the nasopharynx while others need a richer nutritional environment to flourish and so are most often associated with invasive disease. This information may be of use when considering which capsules types to target in future vaccines.

Objective\ud To document trends in invasive pneumococcal disease (IPD) in a central hospital in Malawi during the period of national scale-up of antiretroviral therapy (ART) and cotrimoxazole prophylaxis.\ud \ud Methods\ud Between 1 January 2000 and 31 December 2009 almost 100,000 blood cultures and 40,000 cerebrospinal fluid (CSF) cultures were obtained from adults and children admitted to the Queen Elizabeth Central Hospital, Blantyre, Malawi with suspected severe bacterial infection.\ud \ud Results\ud 4,445 pneumococcal isolates were obtained over the 10 year period. 1,837 were from children: 885 (19.9%) from blood and 952 (21.4%) from CSF. 2,608 were from adults: 1,813 (40.8%) from blood and 795 (17.9%) from CSF. At the start of the surveillance period cotrimoxazole resistance was 73.8% and at the end was 92.6%. Multidrug resistance (MDR) was present in almost one third of isolates and was constant over time. Free ART was introduced in Malawi in 2004. From 2005 onwards there was a decline in invasive pneumococcal infections with a negative correlation between ART scale-up and the decline in IPD (Pearson's correlation r = −0.91; p

Background Epidemiological studies suggest that inhalation of carbonaceous particulate matter from biomass combustion increases susceptibility to bacterial pneumonia. In vitro studies report that phagocytosis of carbon black by alveolar macrophages (AM) impairs killing of Streptococcus pneumoniae. We have previously reported high levels of black carbon in AM from biomass smoke-exposed children and adults. We therefore aimed to use a mouse model to test the hypothesis that high levels of carbon loading of AM in vivo increases susceptibility to pneumococcal pneumonia. Methods Female outbred mice were treated with either intranasal phosphate buffered saline (PBS) or ultrafine carbon black (UF-CB in PBS; 500 μg on day 1 and day 4), and then infected with S. pneumoniae strain D39 on day 5. Survival was assessed over 72 h. The effect of UF-CB on AM carbon loading, airway inflammation, and a urinary marker of pulmonary oxidative stress was assessed in uninfected animals. Results Instillation of UF-CB in mice resulted a pattern of AM carbon loading similar to that of biomass-smoke exposed humans. In uninfected animals, UF-CB treated animals had increased urinary 8-oxodG (P = 0.055), and an increased airway neutrophil differential count (P < 0.01). All PBS-treated mice died within 72 h after infection with S. pneumoniae, whereas morbidity and mortality after infection was reduced in UF-CB treated animals (median survival 48 h vs. 30 h, P < 0.001). At 24 hr post-infection, UF-CB treated mice had lower lung and the blood S. pneumoniae colony forming unit counts, and lower airway levels of keratinocyte-derived chemokine/growth-related oncogene (KC/GRO), and interferon gamma. Conclusion Acute high level loading of AM with ultrafine carbon black particles per se does not increase the susceptibility of mice to pneumococcal infection in vivo.

Streptococcus pneumoniaeis the most common bacterial cause of community-acquired meningitis worldwide. Despite optimal antibiotic therapy and supportive care, the mortality of this condition remains very high at 20–30% in the developed world and over 60% in under-resourced hospitals. In developed countries, approximately half of the survivors suffer intellectual impairment, hearing loss, or other neurological damage. There is an urgent need for more information about the mechanisms of brain damage and death in pneumococcal meningitis so that new treatments can be designed. Using proteomic techniques and bioinformatics, the protein content of cerebrospinal fluid can be examined in great detail. Animal models have added greatly to our knowledge of possible mechanisms and shown that hippocampal apoptosis and cortical necrosis are distinct mechanisms of neuronal death. The contribution of these pathways to human disease is unknown. Using proteomic techniques, neuronal death pathways could be described in CSF samples. This information could lead to the design of novel therapies to minimize brain damage and lower mortality. This minireview will summarize the known pathogenesis of meningitis, and current gaps in knowledge, that could be filled by proteomic analysis.

Background\ud Streptococcus pneumoniae is the leading cause of community-acquired pneumonia in all ages, with the greatest incidence occurring in children, the elderly and HIV-infected individuals. HIV-infected adults are at least 25-fold at risk of bacterial pneumonia compared to age-matched HIV-uninfected individuals, but the immunological mechanisms for this risk are still unclear. Alveolar macrophages (AMs) are the major sentinel phagocytic population found in the airway responsible for the early clearance and control of respiratory pathogens. I hypothesise that HIV infection is associated with impaired airway alveolar phagocyte killing function, leading to survival and propagation of pneumococci.\ud \ud Methods\ud Three groups of participants aged between 18 to 60 years were recruited, consisting of asymptomatic HIV-uninfected adults, HIV-infected adults on shortterm or long-term antiretroviral therapy (ART) residing in Blantyre, Malawi. Lower airway samples (bronchoalveolar lavage fluid) were collected from the participants and used to investigate airway mediated defence immunity against S. pneumoniae in an ex vivo model. The airway cells were infected with pneumococci (serotype 3 strain) and the number of alveolar phagocytes associated with bacteria were measured and phenotyped using flow cytometry. Intracellular and extracellular bacterial killing was measured using quantitative culture in a gentamicin protection assay (GPA) and bacterial outgrowth assay, respectively. Confocal microscopy was used to visualise pneumococci within AMs following infection.\ud \ud Results\ud Alveolar macrophages were the principal phagocytic cells associated with IgGopsonised pneumococcal-ST3 following ex vivo infection, irrespective of HIV status. AM from HIV-infected adults did not exhibit impaired binding and internalisation of IgG-opsonised pneumococcal-ST3, irrespective of ART duration. Furthermore, airway cell-mediated control of extracellular S. pneumoniae outgrowth during early infection was also not impaired in HIV-infected adults on ART. However, HIV-infected adults on ART demonstrated reduced AMs-mediated intracellular killing capacity of S. pneumoniae during the late killing phase (24-hours post infection) with pneumococci persisting in CD206+AMs. The presence of extracellular S. pneumoniae 24-hours post infection in a bacterial outgrowth assay was propagated by intracellular bacterial persistence.\ud \ud Conclusion\ud In summary, the data from this thesis demonstrates that AMs from HIV-infected adults on ART have impaired late bacterial killing capacity, leading to intracellular bacterial persistence and propagation of extracellular pneumococcal outgrowth. Persistence of the pneumococci within CD206+AMs could serve as a reservoir for pneumococcal infection in the lung, potentially contributing to increased susceptibility to pneumococcal pneumonia in HIV-infected individuals.

Introduction \ud Streptococcus pneumoniae (or pneumococcus) is a common commensal (coloniser) of the human upper respiratory tract. Colonisation is likely a prerequisite for respiratory tract infections and invasive pneumococcal disease. Colonisation also has a significant role in the horizontal spread of this pathogen within communities, but paradoxically could also lead to boosting of the host’s immune system. We use the unique experimental human pneumococcal challenge (EHPC) model to study pneumococcal transmission and colonisation in healthy adults. This novel study design allows us to investigate bacteriological and immune factors associated with colonisation and to examine the density and duration of colonisation episodes.\ud Project Aims\ud 1) Investigation of the transmission dynamics of Streptococcus pneumoniae. Can the hands can be a vector for transmission of S. pneumoniae into the nasopharynx, leading to colonisation? Does concurrent asymptomatic viral infection affect transmission? \ud 2) Investigation of the propensity of two pneumococcal serotypes to cause experimental pneumococcal colonisation, to improve the generalisability of the model and to investigate if immunological responses to serotype 6B are similar to other serotypes. We also wanted to investigate if colonisation is an asymptomatic process in healthy adults? How do the host’s polysaccharide specific antibody responses affect colonisation?\ud Main findings \ud Using our unique controlled human pneumococcal challenge model, we have demonstrated the viability of transmission of pneumococcus from the hand into the nasopharynx, leading to colonisation. We were unable to investigate the relationship between colonisation acquisition and concurrent viral infection due to the absence of viral infection in our participants. The data presented in this thesis showed that the experimental human pneumococcal carriage model can successfully investigate transmission dynamics of pneumococcus. We also demonstrated the varying propensity of two pneumococcal serotypes, 23F and 15B to experimentally colonise the nasopharynx of healthy adults. Nasopharyngeal colonisation was shown not to cause nasal symptoms; however, the data suggested that colonisation may cause a cough in healthy adults. No relationship was found between the level of serum IgG to 15B capsular polysaccharide at screening and colonisation outcome after intranasal inoculation. Nasopharyngeal colonisation with 15B was however, found to boost polysaccharide specific immunity; colonisation positive participants had a significant increase in serum IgG levels to 15B capsular PS.\ud Implications \ud Data presented in this thesis suggest that good hand hygiene practices, already known to reduce enteric bacterial and viral disease, may also prevent the spread of pneumococcus which is thought to be spread primarily through aerosolisation. Results support epidemiological studies which have shown the varying propensity of different pneumococcal serotypes to cause colonisation. We can build upon this work by investigating serotypes in vitro and in vivo to understand bacterial factors that impact the pneumococcus’ ability to colonise the nasopharynx in humans. The EHPC model will be useful in further studies to better understand the dynamics and drivers of pneumococcal transmission, bacterial factors which support successful colonisation and host responses to pneumococcal exposure and colonisation.

Live Attenuated Influenza Vaccine (LAIV) is used in immunisation campaigns but may alter the dynamics of naturally occurring nasal colonisation by Streptococcus pneumoniae (Spn), a common human pathogen. We tested how the attenuated influenza viruses contained in the vaccine and Spn interact in the host’s nasopharynx using for the first time an Experimental Human Pneumococcal Challenge model (EHPC) with multiple live pathogens: LAIV and Spn of serotype 6B. Two double blinded randomised clinical trials represented two scenarios of controlled co-infection: 1) Antecedent LAIV administration followed by nasopharyngeal Spn inoculation or 2) Concurrent LAIV administration during established Spn colonisation, separated by a 3 day interval. We validated non-invasive micro-sampling techniques for mucosal immunity analysis by comparing reliability and reproducibility of available methods. Absorptive matrices and nasal curettes were established as the preferred techniques to investigate lining fluid and immune cells in the nasal mucosa. In addition, we collected nasal wash, BAL and serum from healthy adults to investigate immune cell recruitment, cytokine and influenza-specific antibody responses using flow cytometer, human cytokine 30-plex panel and ELISA analysis. Here, we showed that LAIV-induced inflammation in the nasopharynx was associated with Spn colonisation. Immune responses to Spn and to the attenuated influenza virus were impaired by LAIV, reducing chemoattractant cytokines, recruitment of monocytes, and activation of T-cells and neutrophils. In the lung, our results demonstrated that LAIV induces inflammatory cytokines produced by T-cells and that tissue-resident memory T-cells have an important role in producing specific cytokines against the attenuated influenza virus. In short, LAIV was shown to be immunogenic in healthy adults, but less in Spn colonised individuals, highlighting the significance of nasal microbiota when developing vaccines and assessing its efficacy.