The human lung immune responses to nasopharyngeal pneumococcal colonisation in healthy adults

Pneumococcal pneumonia burden remains high globally, affecting the youngest, the oldest and immunocompromised individuals worldwide. Colonisation of the human nasopharynx with S. pneumoniae is a pre-requisite for the development of pneumococcal disease and the primary reservoir for transmission. Paradoxically, it is also the main source of naturally acquired immunity. Currently, there is limited knowledge on how nasopharyngeal pneumococcal colonisation impacts on the lung immune responses in humans. Experimental human pneumococcal colonisation offers a safe way to study the dynamics between a known start-point colonisation episode and the lung immune responses. The data presented in this thesis show that colonisation of the nasopharynx with S. pneumoniae is an immunising event for the human lung, affecting both the innate and adaptive arm of immunity. Alveolar macrophage activation and function were altered post colonisation, leading to increased opsonophagocytic capacity that persisted for up to three months post the challenge. The overall skewed CD4+ Th-1 responses observed in the lung indicate that CD4+ T cells may prime alveolar macrophage through IFN-γ secretion. In vitro stimulation of lung lymphocytes with pneumococcus suggest that TCR-γδ T cells can stand as an additional source of IFN-γ in vivo. As described before, pneumococcal colonisation seeded the human lung with cognate, IL-17 secreting, CD4+ T cells- the only observed memory population amongst lung T cells. Antibody levels against the capsule of the challenge strain were also elevated post colonisation. Pneumococcal cells found in the lung days to weeks after clearance of nasal colonisation suggest that they may be the stimulus for the observed enhanced lung immunity. Collectively, the data presented here support the use of respiratory tract as route for effective pneumococcal vaccination. On the other hand, they emphasize that heavily colonised individuals with defective alveolar macrophage function or elderly due to lack of colonisation are at increased risk to develop pneumonia

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

Single use and conventional bronchoscopes for Broncho alveolar lavage (BAL) in research: a comparative study (NCT 02515591)

Background Broncho alveolar lavage (BAL) is widely used for investigative research to study innate, cellular and humoral immune responses, and in early phase drug trials. Conventional (multiple use) flexible bronchoscopes have time and monetary costs associated with cleaning, and carries a small risk of cross infection. Single use bronchoscopes may provide an alternative, but have not been evaluated in this context. Methods Healthy volunteers underwent bronchoscopy at a day-case clinical research unit using the Ambu® aScopeTM single-use flexible intubation bronchoscope. Broncho alveolar lavage was performed from a sub segmental bronchus within the right middle lobe; a total of 200 ml of warmed normal saline was instilled then aspirated using handheld suction. BAL volume yield, cell yield and viability were recorded. Results Ten volunteers, (mean age 23 years, six male) participated. Bronchoscopies were carried out by one of two senior bronchoscopists, experienced in the technique of obtaining BAL for research purposes. The results were compared to 50 (mean age 23, 14 male) procedures performed using the conventional scope by the same two bronchoscopists. The total volume yield was significantly higher in the disposable group median 152 ml (IQR 141–166 ml) as compared to conventional 124 ml (110–135 ml), p = <0.01. The total cell yield and viability were similar in both groups, with no significant differences. Conclusions With single use bronchoscopes, we achieved a larger BAL volume yield than conventional bronchoscopes, with comparable cell yield and viability. Better volume yields can potentially reduce post procedure side effects such as pleuritic chest pain and cough. The risk of cross infection can be eliminated, providing reassurance to researchers and participants. Reduced maintenance requirements can be cost effective. These could potentially be used for early phase drug development studies. Trial registration This trial was registered prospectively in July 2015 with the National Clinical Trials register, with the following registration number assigned: NCT 02515591

Airway-resident T cells from unexposed individuals cross-recognize SARS-CoV-2.

T cells can contribute to clearance of respiratory viruses that cause acute-resolving infections such as SARS-CoV-2, helping to provide long-lived protection against disease. Recent studies have suggested an additional role for T cells in resisting overt infection: pre-existing cross-reactive responses were preferentially enriched in healthcare workers who had abortive infections1, and in household contacts protected from infection2. We hypothesize that such early viral control would require pre-existing cross-reactive memory T cells already resident at the site of infection; such airway-resident responses have been shown to be critical for mediating protection after intranasal vaccination in a murine model of SARS-CoV3. Bronchoalveolar lavage samples from the lower respiratory tract of healthy donors obtained before the COVID-19 pandemic revealed airway-resident, SARS-CoV-2-cross-reactive T cells, which correlated with the strength of human seasonal coronavirus immunity. We therefore demonstrate the potential to harness functional airway-resident SARS-CoV-2-reactive T cells in next-generation mucosal vaccines

Thirteen-Valent Pneumococcal Conjugate Vaccine–Induced Immunoglobulin G (IgG) Responses in Serum Associated With Serotype-Specific IgG in the Lung

Pneumococcal conjugate vaccine (PCV) efficacy is lower for noninvasive pneumonia than invasive disease. In this study, participants were immunized with 13-valent PCV (PCV13) or hepatitis A vaccine (control). Bronchoalveolar lavage samples were taken between 2 and 6 months and serum at 4 and 7 weeks postvaccination. In the lung, anti-capsular immunoglobulin G (IgG) levels were higher in the PCV13 group compared to controls for all serotypes, except 3 and 6B. Systemically, IgG levels were elevated in the PCV13 group at 4 weeks for all serotypes, except serotype 3. IgG in bronchoalveolar lavage and serum positively correlated for nearly all serotypes. PCV13 shows poor immunogenicity to serotype 3, implying lack of protective efficacy

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

Angiogenesis in cancer of unknown primary: clinicopathological study of CD34, VEGF and TSP-1

BACKGROUND: Cancer of unknown primary remains a mallignancy of elusive biology and grim prognosis that lacks effective therapeutic options. We investigated angiogenesis in cancer of unknown primary to expand our knowledge on the biology of these tumors and identify potential therapeutic targets. METHODS: Paraffin embedded archival material from 81 patients diagnosed with CUP was used. Tumor histology was adenocarcinoma (77%), undifferentiated carcinoma (18%) and squamous cell carcinoma (5%). The tissue expression of CD34, VEGF and TSP-1 was assessed immunohistochemically by use of specific monoclonal antibodies and was analyzed against clinicopathological data. RESULTS: VEGF expression was detected in all cases and was strong in 83%. Stromal expression of TSP-1 was seen in 80% of cases and was strong in 20%. The expression of both proteins was not associated with any clinical or pathological parameters. Tumor MVD was higher in tumors classified as unfavorable compared to more favorable and was positively associated with VEGF and negatively with TSP-1. CONCLUSION: Angiogenesis is very active and expression of VEGF is almost universal in cancers of unknown primary. These findings support the clinical investigation of VEGF targeted therapy in this clinical setting

Serotype 3 Experimental Human Pneumococcal Challenge (EHPC) study protocol: dose ranging and reproducibility in a healthy volunteer population (challenge 3)

Introduction: Since the introduction of pneumococcal conjugate vaccines, pneumococcal disease rates have declined for many vaccine-type serotypes. However, serotype 3 (SPN3) continues to cause significant disease and is identified in colonisation epidemiological studies as one of the top circulating serotypes in adults in the UK. Consequently, new vaccines that provide greater protection against SPN3 colonisation/carriage are urgently needed. The Experimental Human Pneumococcal Challenge (EHPC) model is a unique method of determining pneumococcal colonisation rates, understanding acquired immunity, and testing vaccines in a cost-effective manner. To enhance the development of effective pneumococcal vaccines against SPN3, we aim to develop a new relevant and safe SPN3 EHPC model with high attack rates which could be used to test vaccines using small sample size. Methods and analysis: This is a human challenge study to establish a new SPN3 EHPC model, consisting of two parts. In the dose-ranging/safety study, cohorts of 10 healthy participants will be challenged with escalating doses of SPN3. If first challenge does not lead into colonisation, participants will receive a second challenge 2 weeks after. Experimental nasopharyngeal (NP) colonisation will be determined using nasal wash sampling. Using the dose that results in ≥50% of participants being colonised, with a high safety profile, we will complete the cohort with another 33 participants to check for reproducibility of the colonisation rate. The primary outcome of this study is to determine the optimal SPN3 dose and inoculation regime to establish the highest rates of NP colonisation in healthy adults. Secondary outcomes include determining density and duration of experimental SPN3 NP colonisation and characterising mucosal and systemic immune responses to SPN3 challenge. Ethics and dissemination: This study is approved by the NHS Research and Ethics Committee (reference 22/NW/0051). Findings will be published in peer-reviewed journals and reports will be made available to participants

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