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

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

A Randomized Controlled Clinical Trial of Nasal Immunization with Live Virulence Attenuated Streptococcus pneumoniae Strains Using Human Infection Challenge.

Rationale: Pneumococcal pneumonia remains a global health problem. Pneumococcal colonization increases local and systemic protective immunity, suggesting that nasal administration of live attenuated Streptococcus pneumoniae (Spn) strains could help prevent infections. Objectives: We used a controlled human infection model to investigate whether nasopharyngeal colonization with attenuated S. pneumoniae strains protected against recolonization with wild-type (WT) Spn (SpnWT). Methods: Healthy adults aged 18-50 years were randomized (1:1:1:1) for nasal administration twice (at a 2-wk interval) with saline solution, WT Spn6B (BHN418), or one of two genetically modified Spn6B strains, SpnA1 (Δfhs/piaA) or SpnA3 (ΔproABC/piaA) (Stage I). After 6 months, participants were challenged with SpnWT to assess protection against the homologous serotype (Stage II). Measurements and Main Results: 125 participants completed both study stages per intention to treat. No serious adverse events were reported. In Stage I, colonization rates were similar among groups: SpnWT, 58.1% (18 of 31); SpnA1, 60% (18 of 30); and SpnA3, 59.4% (19 of 32). Anti-Spn nasal IgG levels after colonization were similar in all groups, whereas serum IgG responses were higher in the SpnWT and SpnA1 groups than in the SpnA3 group. In colonized individuals, increases in IgG responses were identified against 197 Spn protein antigens and serotype 6 capsular polysaccharide using a pangenome array. Participants given SpnWT or SpnA1 in Stage I were partially protected against homologous challenge with SpnWT (29% and 30% recolonization rates, respectively) at stage II, whereas those exposed to SpnA3 achieved a recolonization rate similar to that in the control group (50% vs. 47%, respectively). Conclusions: Nasal colonization with genetically modified live attenuated Spn was safe and induced protection against recolonization, suggesting that nasal administration of live attenuated Spn could be an effective strategy for preventing pneumococcal infections. Clinical trial registered with the ISRCTN registry (ISRCTN22467293).

Human Infection Challenge with Serotype 3 Pneumococcus.

Rationale: Streptococcus 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) programs, 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. Objectives: To establish SPN3's ability to colonize the nasopharynx using different inoculum clades and doses, and the safety of an SPN3 challenge model. Methods: In a human challenge study involving three well-characterized and antibiotic-sensitive SPN3 isolates (PFESP306 [clade Ia], PFESP231 [no clade], and PFESP505 [clade II]), inoculum doses (10,000, 20,000, 80,000, and 160,000 cfu/100 µl) were escalated until maximal colonization rates were achieved, with concurrent acceptable safety. Measurement and Main Results: Presence and density of experimental SPN3 nasopharyngeal colonization in nasal wash samples, assessed using microbiological culture and molecular methods, on Days 2, 7, and 14 postinoculation. A total of 96 healthy participants (median age 21, interquartile range 19-25) were inoculated (n = 6-10 per dose group, 10 groups). Colonization rates ranged from 30.0-70.0% varying with dose and isolate. 30.0% (29/96) reported mild symptoms (82.8% [24/29] developed a sore throat); one developed otitis media requiring antibiotics. No serious adverse events occurred. Conclusions: An SPN3 human challenge model is feasible and safe with comparable carriage rates to an established Serotype 6B human challenge model. SPN3 carriage may cause mild upper respiratory symptoms.

Targeted Transcriptomic Screen of Pneumococcal Genes Expressed during Murine and Human Infection.

The advent of pneumococcal conjugate vaccines led to the near disappearance of most of the included serotypes in high-income settings but also the rise of nonvaccine-type colonization and disease. Alternative strategies, using genetically conserved proteins as antigens, have been evaluated preclinically and clinically for years, so far unsuccessfully. One possible explanation for the failure of these efforts is that the choice of antigens may not have been sufficiently guided by an understanding of the gene expression pattern in the context of infection. Here, we present a targeted transcriptomic analysis of 160 pneumococcal genes encoding bacterial surface-exposed proteins in mouse models, human colonization, and human meningitis. We present the overlap of these different transcriptomic profiles. We identify two bacterial genes that are highly expressed in the context of mouse and human infection: SP_0282, an IID component of a mannose phosphotransferase system (PTS), and SP_1739, encoding RNase Y. We show that these two proteins can confer protection against pneumococcal nasopharyngeal colonization and intraperitoneal challenge in a murine model and generate opsonophagocytic antibodies. This study emphasizes and confirms the importance of studies of pneumococcal gene expression of bacterial surface proteins during human infection and colonization and may pave the way for the selection of a protein-based vaccine candidate.

Experimental Human Pneumococcal Colonization in Older Adults Is Feasible and Safe, Not Immunogenic.

Rationale: Pneumococcal colonization is key to the pathogenesis of invasive disease but is also immunogenic in young adults, protecting against recolonization. Colonization is rarely detected in older adults, despite high rates of pneumococcal disease.Objectives: To establish experimental human pneumococcal colonization in healthy adults aged 50-84 years, to measure the immune response to pneumococcal challenge, and to assess the protective effect of prior colonization against autologous strain rechallenge.Methods: Sixty-four participants were inoculated with Streptococcus pneumoniae (serotype 6B; 80,000 cfu in each nostril). Colonization was determined by bacterial culture of nasal wash, and humoral immune responses were assessed by anticapsular and antiprotein IgG concentrations.Measurements and Main Results: Experimental colonization was established in 39% of participants (25/64) with no adverse events. Colonization 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 colonization. Colonization did not confer serotype-specific immune boosting, with a geometric mean titer (95% confidence interval) of 2.7 µg/ml (1.9-3.8) before the challenge versus 3.0 (1.9-4.7) 4 weeks after colonization (P = 0.53). Furthermore, pneumococcal challenge without colonization led to a drop in specific antibody concentrations from 2.8 µg/ml (2.0-3.9) to 2.2 µg/ml (1.6-3.0) after the challenge (P = 0.006). Antiprotein antibody concentrations increased after successful colonization. Rechallenge with the same strain after a median of 8.5 months (interquartile range, 6.7-10.1) led to recolonization in 5/16 (31%).Conclusions: In older adults, experimental pneumococcal colonization is feasible and safe but demonstrates different immunological outcomes compared with younger adults in previous studies.

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 Streptococcus 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 BAL from healthy pneumococcal-challenged participants aged 18-49 years. Confocal microscopy and 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: AMs from Spn-colonized individuals exhibited increased opsonophagocytosis to pneumococcus (11.4% median increase) for approximately 3 months after experimental pneumococcal colonization. AMs also had increased responses against other bacterial pathogens. Pneumococcal DNA detected in the BAL samples of Spn-colonized individuals 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 AMs, leading to brisker responsiveness to both pneumococcus and unrelated bacterial pathogens. The relative abundance of AMs in the alveolar spaces, alongside their potential for nonspecific protection, render them an attractive target for novel vaccines.

Intrapulmonary Pharmacokinetics of Cefepime and Enmetazobactam in Healthy Volunteers: Towards New Treatments for Nosocomial Pneumonia.

Cefepime-enmetazobactam is a novel ß-lactam-ß-lactamase inhibitor combination with broad-spectrum antimicrobial activity against a range of multidrug-resistant Enterobacteriaceae This agent is being developed for a range of serious hospital infections. An understanding of the extent of partitioning of ß-lactam-ß-lactamase inhibitor combinations into the human lung is required to better understand the potential role of cefepime-enmetazobactam for the treatment of nosocomial pneumonia. A total of 20 healthy volunteers were used to study the intrapulmonary pharmacokinetics of a regimen of 2 g cefepime-1 g enmetazobactam every 8 h intravenously (2 g/1 g q8h i.v.). Each volunteer contributed multiple plasma samples and a single epithelial lining fluid (ELF) sample, obtained by bronchoalveolar lavage. Concentrations of cefepime and enmetazobactam were quantified using liquid chromatography-tandem mass spectrometry. The pharmacokinetic data were modeled using a population methodology, and Monte Carlo simulations were performed to assess the attainment of pharmacodynamic targets defined in preclinical models. The concentration-time profiles of both agents in plasma and ELF were similar. The mean ± standard deviation percentage of partitioning of total drug concentrations of cefepime and enmetazobactam between plasma and ELF was 60.59% ± 28.62% and 53.03% ± 21.05%, respectively. Using pharmacodynamic targets for cefepime of greater than the MIC and free enmetazobactam concentrations of >2 mg/liter in ELF of 20% of the dosing interval, a regimen of cefepime-enmetazobactam of 2 g/0.5 g q8h i.v. infused over 2 h resulted in a probability of target attainment of ≥90% for Enterobacteriaceae with cefepime-enmetazobactam MICs of ≤8 mg/liter. This result provides a rationale to further consider cefepime-enmetazobactam for the treatment of nosocomial pneumonia caused by multidrug-resistant Enterobacteriaceae.

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 .

Tetanus-diphtheria vaccine can prime SARS-CoV-2 cross-reactive T cells.

Vaccines containing tetanus-diphtheria antigens have been postulated to induce cross-reactive immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which could protect against coronavirus disease (COVID-19). In this work, we investigated the capacity of Tetanus-diphtheria (Td) vaccine to prime existing T cell immunity to SARS-CoV-2. To that end, we first collected known SARS-CoV-2 specific CD8+ T cell epitopes targeted during the course of SARS-CoV-2 infection in humans and identified as potentially cross-reactive with Td vaccine those sharing similarity with tetanus-diphtheria vaccine antigens, as judged by Levenshtein edit distances (≤ 20% edits per epitope sequence). As a result, we selected 25 potentially cross-reactive SARS-CoV-2 specific CD8+ T cell epitopes with high population coverage that were assembled into a synthetic peptide pool (TDX pool). Using peripheral blood mononuclear cells, we first determined by intracellular IFNγ staining assays existing CD8+ T cell recall responses to the TDX pool and to other peptide pools, including overlapping peptide pools covering SARS-CoV-2 Spike protein and Nucleocapsid phosphoprotein (NP). In the studied subjects, CD8+ T cell recall responses to Spike and TDX peptide pools were dominant and comparable, while recall responses to NP peptide pool were less frequent and weaker. Subsequently, we studied responses to the same peptides using antigen-inexperienced naive T cells primed/stimulated in vitro with Td vaccine. Priming stimulations were carried out by co-culturing naive T cells with autologous irradiated peripheral mononuclear cells in the presence of Td vaccine, IL-2, IL-7 and IL-15. Interestingly, naive CD8+ T cells stimulated/primed with Td vaccine responded strongly and specifically to the TDX pool, not to other SARS-CoV-2 peptide pools. Finally, we show that Td-immunization of C57BL/6J mice elicited T cells cross-reactive with the TDX pool. Collectively, our findings support that tetanus-diphtheria vaccines can prime SARS-CoV-2 cross-reactive T cells and likely contribute to shape the T cell responses to the virus.

Experimental pneumococcal carriage in people living with HIV in Malawi: the first controlled human infection model in a key at-risk population.

Background: As well as suffering a high burden of pneumococcal disease people living with HIV (PLHIV) may contribute to community transmission in sub-Saharan African (sSA) settings. Pneumococcal vaccination is not currently offered to PLHIV in sSA but may prevent disease and reduce transmission. More evidence of vaccine effectiveness against carriage in PLHIV is needed. An Experimental Human Pneumococcal Carriage model (EHPC) has been safely and acceptably used in healthy adults in Malawi to evaluate pneumococcal vaccines against carriage and to identify immune correlates of protection from carriage. This study will establish the same model in PLHIV and will be the first controlled human infection model (CHIM) in this key population. Methods: Healthy participants with and without HIV will be inoculated intranasally with Streptococcus pneumoniae serotype 6B. Sequential cohorts will be challenged with increasing doses to determine the optimal safe challenge dose to establish experimental carriage. Nasal fluid, nasal mucosal, and blood samples will be taken before inoculation and on days 2, 7, 14, and 21 following inoculation to measure pneumococcal carriage density and identify immune correlates of protection from carriage. The vast majority of natural pneumococcal carriage events in PLHIV do not result in invasive disease and no invasive disease is expected in this study. However, robust participant safety monitoring is designed to identify signs of invasive disease early should they develop, and to implement treatment immediately. Participants will complete a Likert-style questionnaire at study-end to establish acceptability. Interpretations: We expect the EHPC model to be safely and acceptably implemented in PLHIV. The CHIM can then be used to accelerate pneumococcal vaccine evaluations in this population, and an evidence-based pneumococcal vaccination policy for PLHIV in sSA.

Inflammation of the nasal mucosa is associated with susceptibility to experimental pneumococcal challenge in older adults.

Streptococcus pneumoniae colonization in the upper respiratory tract is linked to pneumococcal disease development, predominantly affecting young children and older adults. As the global population ages and comorbidities increase, there is a heightened concern about this infection. We investigated the immunological responses of older adults to pneumococcal-controlled human infection by analyzing the cellular composition and gene expression in the nasal mucosa. Our comparative analysis with data from a concurrent study in younger adults revealed distinct gene expression patterns in older individuals susceptible to colonization, highlighted by neutrophil activation and elevated levels of CXCL9 and CXCL10. Unlike younger adults challenged with pneumococcus, older adults did not show recruitment of monocytes into the nasal mucosa following nasal colonization. However, older adults who were protected from colonization showed increased degranulation of cluster of differentiation 8+ T cells, both before and after pneumococcal challenge. These findings suggest age-associated cellular changes, in particular enhanced mucosal inflammation, that may predispose older adults to pneumococcal colonization.

Antibody and B-cell Immune Responses Against Bordetella Pertussis Following Infection and Immunization.

Neither immunization nor recovery from natural infection provides life-long protection against Bordetella pertussis. Replacement of a whole-cell pertussis (wP) vaccine with an acellular pertussis (aP) vaccine, mutations in B. pertussis strains, and better diagnostic techniques, contribute to resurgence of number of cases especially in young infants. Development of new immunization strategies relies on a comprehensive understanding of immune system responses to infection and immunization and how triggering these immune components would ensure protective immunity. In this review, we assess how B cells, and their secretory products, antibodies, respond to B. pertussis infection, current and novel vaccines and highlight similarities and differences in these responses. We first focus on antibody-mediated immunity. We discuss antibody (sub)classes, elaborate on antibody avidity, ability to neutralize pertussis toxin, and summarize different effector functions, i.e. ability to activate complement, promote phagocytosis and activate NK cells. We then discuss challenges and opportunities in studying B-cell immunity. We highlight shared and unique aspects of B-cell and plasma cell responses to infection and immunization, and discuss how responses to novel immunization strategies better resemble those triggered by a natural infection (i.e., by triggering responses in mucosa and production of IgA). With this comprehensive review, we aim to shed some new light on the role of B cells and antibodies in the pertussis immunity to guide new vaccine development.

Respiratory mucosal immune memory to SARS-CoV-2 after infection and vaccination.

Respiratory mucosal immunity induced by vaccination is vital for protection from coronavirus infection in animal models. In humans, the capacity of peripheral vaccination to generate sustained immunity in the lung mucosa, and how this is influenced by prior SARS-CoV-2 infection, is unknown. Here we show using bronchoalveolar lavage samples that donors with history of both infection and vaccination have more airway mucosal SARS-CoV-2 antibodies and memory B cells than those only vaccinated. Infection also induces populations of airway spike-specific memory CD4+ and CD8+ T cells that are not expanded by vaccination alone. Airway mucosal T cells induced by infection have a distinct hierarchy of antigen specificity compared to the periphery. Spike-specific T cells persist in the lung mucosa for 7 months after the last immunising event. Thus, peripheral vaccination alone does not appear to induce durable lung mucosal immunity against SARS-CoV-2, supporting an argument for the need for vaccines targeting the airways.

Detection of SARS-CoV-2 infection by saliva and nasopharyngeal sampling in frontline healthcare workers: An observational cohort study.

BACKGROUND: The SARS-CoV-2 pandemic has caused an unprecedented strain on healthcare systems worldwide, including the United Kingdom National Health Service (NHS). We conducted an observational cohort study of SARS-CoV-2 infection in frontline healthcare workers (HCW) working in an acute NHS Trust during the first wave of the pandemic, to answer emerging questions surrounding SARS-CoV-2 infection, diagnosis, transmission and control. METHODS: Using self-collected weekly saliva and twice weekly combined oropharyngeal/nasopharyngeal (OP/NP) samples, in addition to self-assessed symptom profiles and isolation behaviours, we retrospectively compared SARS-CoV-2 detection by RT-qPCR of saliva and OP/NP samples. We report the association with contemporaneous symptoms and isolation behaviour. RESULTS: Over a 12-week period from 30th March 2020, 40·0% (n = 34/85, 95% confidence interval 31·3-51·8%) HCW had evidence of SARS-CoV-2 infection by surveillance OP/NP swab and/or saliva sample. Symptoms were reported by 47·1% (n = 40) and self-isolation by 25·9% (n = 22) participants. Only 44.1% (n = 15/34) participants with SARS-CoV-2 infection reported any symptoms within 14 days of a positive result and only 29·4% (n = 10/34) reported self-isolation periods. Overall agreement between paired saliva and OP/NP swabs was 93·4% (n = 211/226 pairs) but rates of positive concordance were low. In paired samples with at least one positive result, 35·0% (n = 7/20) were positive exclusively by OP/NP swab, 40·0% (n = 8/20) exclusively by saliva and in only 25·0% (n = 5/20) were the OP/NP and saliva result both positive. CONCLUSIONS: HCW are a potential source of SARS-CoV-2 transmission in hospitals and symptom screening will identify the minority of infections. Without routine asymptomatic SARS-CoV-2 screening, it is likely that HCW with SARS-CoV-2 infection would continue to attend work. Saliva, in addition to OP/NP swab testing, facilitated ascertainment of symptomatic and asymptomatic SARS-CoV-2 infections. Combined saliva and OP/NP swab sampling would improve detection of SARS-CoV-2 for surveillance and is recommended for a high sensitivity strategy.

Influence of sex, season and environmental air quality on experimental human pneumococcal carriage acquisition: a retrospective cohort analysis.

Streptococcus pneumoniae (pneumococcus) is the most commonly identified bacterial cause of pneumonia and the leading infectious cause of death in children under 5 years of age worldwide. Pneumococcal disease follows a seasonal pattern with increased incidence during winter. Pneumonia burden is also associated with poor air quality. Nasopharyngeal carriage of the bacterium is a pre-requisite of invasive disease. We aimed to determine if susceptibility to nasopharyngeal pneumococcal carriage varied by season and which environmental factors might explain such variation. We also evaluated the influence of sex on susceptibility of carriage. We collated data from five studies in which human volunteers underwent intranasal pneumococcal challenge. Generalised linear mixed-effects models were used to identify factors associated with altered risk of carriage acquisition, specifically climate and air-quality data. During 2011-2017, 374 healthy adults were challenged with type 6B pneumococcus. Odds of carriage were significantly lower in males (OR, 0.61; 95% CI, 0.40-0.92; p=0.02), and higher with cooler temperatures (OR, 0.79; 95% CI, 0.63-0.99; p=0.04). Likelihood of carriage was also associated with lower concentrations of local fine particulate matter concentrations (PM2.5) and increased local rainfall. In contrast to epidemiological series, experimental challenge allowed us to test propensity to acquisition during controlled exposures; immunological explanations for sex and climatic differences should be sought.

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. TRIAL REGISTRATION NUMBER: ISRCTN15728847, NCT04974294.

Streptococcus pneumoniae colonization associates with impaired adaptive immune responses against SARS-CoV-2.

BackgroundAlthough recent epidemiological data suggest that pneumococci may contribute to the risk of SARS-CoV-2 disease, cases of coinfection with Streptococcus pneumoniae in patients with coronavirus disease 2019 (COVID-19) during hospitalization have been reported infrequently. This apparent contradiction may be explained by interactions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and pneumococci in the upper airway, resulting in the escape of SARS-CoV-2 from protective host immune responses.MethodsHere, we investigated the relationship of these 2 respiratory pathogens in 2 distinct cohorts of health care workers with asymptomatic or mildly symptomatic SARS-CoV-2 infection identified by systematic screening and patients with moderate to severe disease who presented to the hospital. We assessed the effect of coinfection on host antibody, cellular, and inflammatory responses to the virus.ResultsIn both cohorts, pneumococcal colonization was associated with diminished antiviral immune responses, which primarily affected mucosal IgA levels among individuals with mild or asymptomatic infection and cellular memory responses in infected patients.ConclusionOur findings suggest that S. pneumoniae impair host immunity to SARS-CoV-2 and raise the question of whether pneumococcal carriage also enables immune escape of other respiratory viruses and facilitates reinfection.Trial registrationISRCTN89159899 (FASTER study) and ClinicalTrials.gov NCT03502291 (LAIV study).

CD3γ-independent pathways in TCR-mediated signaling in mature T and iNKT lymphocytes.

Antigen recognition by T-lymphocytes through the T-cell antigen receptor, TCR-CD3, is a central event in the initiation of an immune response. CD3 proteins may have redundant as well as specific contributions to the intracellular propagation of TCR-mediated signals. However, to date, the relative role that each CD3 chain plays in signaling is controversial. In order to examine the roles of CD3γ chain in TCR signaling, we analyzed proximal and distal signaling events in human CD3γ(-/-) primary and Herpesvirus saimiri (HVS)-transformed T cells. Following TCR-CD3 engagement, certain early TCR signaling pathways (ZAP-70, ERK, p38 and mTORC2 phosphorylation, and actin polymerization) were comparable with control HVS-transformed T cells. However, other signaling pathways were affected, such TCRζ phosphorylation, indicating that the CD3γ chain contributes to improve TCR signaling efficiency and survival. On the other hand, CD3γ(-/-) primary invariant NKT cells (iNKT cells) showed a normal expansion in response to alpha-galactosylceramide (α-GalCer) and TCRVß11(bright) iNKT cells were preferentially selected in this in vitro culture system, perhaps as a consequence of selective events in the thymus. Our results collectively indicate that a TCR lacking CD3γ can propagate a number of signals through the remaining invariant chains, likely the homologous CD3δ chain, which replaces it at the mutant TCR.

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.

Experimental Human Challenge Defines Distinct Pneumococcal Kinetic Profiles and Mucosal Responses between Colonized and Non-Colonized Adults.

Colonization of the upper respiratory tract with Streptococcus pneumoniae is the precursor of pneumococcal pneumonia and invasive disease. Following exposure, however, it is unclear which human immune mechanisms determine whether a pathogen will colonize. We used a human challenge model to investigate host-pathogen interactions in the first hours and days following intranasal exposure to Streptococcus pneumoniae Using a novel home sampling method, we measured early immune responses and bacterial density dynamics in the nose and saliva after volunteers were experimentally exposed to pneumococcus. Here, we show that nasal colonization can take up to 24 h to become established. Also, the following two distinct bacterial clearance profiles were associated with protection: nasal clearers with immediate clearance of bacteria in the nose by the activity of pre-existent mucosal neutrophils and saliva clearers with detectable pneumococcus in saliva at 1 h post challenge and delayed clearance mediated by an inflammatory response and increased neutrophil activity 24 h post bacterial encounter. This study describes, for the first time, how colonization with a bacterium is established in humans, signifying that the correlates of protection against pneumococcal colonization, which can be used to inform design and testing of novel vaccine candidates, could be valid for subsets of protected individuals.IMPORTANCE Occurrence of lower respiratory tract infections requires prior colonization of the upper respiratory tract with a pathogen. Most bacterial infection and colonization studies have been performed in murine and in vitro models due to the current invasive sampling methodology of the upper respiratory tract, both of which poorly reflect the complexity of host-pathogen interactions in the human nose. Self-collecting saliva and nasal lining fluid at home is a fast, low-cost, noninvasive, high-frequency sampling platform for continuous monitoring of bacterial encounter at defined time points relative to exposure. Our study demonstrates for the first time that, in humans, there are distinct profiles of pneumococcal colonization kinetics, distinguished by speed of appearance in saliva, local phagocytic function, and acute mucosal inflammatory responses, which may either recruit or activate neutrophils. These data are important for the design and testing of novel vaccine candidates.