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Virulence screens have indicated potential roles during Streptococcus pneumoniae infection for the one-carbon metabolism pathway component Fhs and proline synthesis mediated by ProABC. To define how these metabolic pathways affect S. pneumoniae virulence, we have investigated the phenotypes, transcription, and metabolic profiles of Δ fhs and Δ proABC mutants. S. pneumoniae capsular serotype 6B BHN418 Δ fhs and Δ proABC mutant strains had strongly reduced virulence in mouse sepsis and pneumonia models but could colonize the nasopharynx. Both mutant strains grew normally in complete media but had markedly impaired growth in chemically defined medium, human serum, and human cerebrospinal fluid. The BHN418 Δ proABC strain also had impaired growth under conditions of osmotic and oxidative stress. The virulence role of proABC was strain specific, as the D39 Δ proABC strain could still cause septicemia and grow in serum. Compared to culture in broth, in serum, the BHN418 Δ fhs and Δ proABC strains showed considerable derangement in global gene transcription that affected multiple but different metabolic pathways for each mutant strain. Metabolic data suggested that Δ fhs had an impaired stringent response, and when cultured in sera, BHN418 Δ fhs and Δ proABC were under increased oxidative stress and had altered lipid profiles. Loss of proABC also affected carbohydrate metabolism and the accumulation of peptidoglycan synthesis precursors in the BHN418 but not the D39 background, linking this phenotype to the conditional virulence phenotype. These data identify the S. pneumoniae metabolic functions affected by S. pneumoniae one-carbon metabolism and proline biosynthesis, and the role of these genetic loci for establishing systemic infection.IMPORTANCERapid adaptation to grow within the physiological conditions found in the host environment is an essential but poorly understood virulence requirement for systemic pathogens such as Streptococcus pneumoniae . We have now demonstrated an essential role for the one-carbon metabolism pathway and a conditional role depending on strain background for proline biosynthesis for S. pneumoniae growth in serum or cerebrospinal fluid, and therefore for systemic virulence. RNAseq and metabolomic data demonstrated that the loss of one-carbon metabolism or proline biosynthesis has profound but differing effects on S. pneumoniae metabolism in human serum, identifying the metabolic processes dependent on each pathway during systemic infection. These data provide a more detailed understanding of the adaptations required by systemic bacterial pathogens in order to cause infection and demonstrate that the requirement for some of these adaptations varies between strains from the same species and could therefore underpin strain variations in virulence potential.

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., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Epidemiological studies report the impact of co-infection with pneumococcus and respiratory viruses upon disease rates and outcomes, but their effect on pneumococcal carriage acquisition and bacterial load is scarcely described. Here, we assess this by combining natural viral infection with controlled human pneumococcal infection in 581 healthy adults screened for upper respiratory tract viral infection before intranasal pneumococcal challenge. Across all adults, respiratory syncytial virus (RSV) and rhinovirus asymptomatic infection confer a substantial increase in secondary infection with pneumococcus. RSV also has a major impact on pneumococcal density up to 9 days post challenge. We also study rates and kinetics of bacterial shedding through the nose and oral route in a subset. High levels of pneumococcal colonization density and nasal inflammation are strongly correlated with increased odds of nasal shedding as opposed to cough shedding. Protection against respiratory viral infections and control of pneumococcal density may contribute to preventing pneumococcal disease and reducing bacterial spread., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Tools to evaluate and accelerate tuberculosis (TB) vaccine development are needed to advance global TB control strategies. Validated human infection studies for TB have the potential to facilitate breakthroughs in understanding disease pathogenesis, identify correlates of protection, develop diagnostic tools, and accelerate and de-risk vaccine and drug development. However, key challenges remain for realizing the clinical utility of these models, which require further discussion and alignment among key stakeholders. In March 2023, the Wellcome Trust and the International AIDS Vaccine Initiative convened international experts involved in developing both TB and bacillus Calmette-Guérin (BCG) human infection studies (including mucosal and intradermal challenge routes) to discuss the status of each of the models and the key enablers to move the field forward. This report provides a summary of the presentations and discussion from the meeting. Discussions identified key issues, including demonstrating model validity, to provide confidence for vaccine developers, which may be addressed through demonstration of known vaccine effects (eg, BCG vaccination in specific populations), and by comparing results from field efficacy and human infection studies. The workshop underscored the importance of establishing safe and acceptable studies in high-burden settings, and the need to validate >1 model to allow for different scientific questions to be addressed as well as to provide confidence to vaccine developers and regulators around use of human infection study data in vaccine development and licensure pathways., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. C. L. W., S. B., N. R. S., and C. N. L. are employees of Wellcome Trust. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Pneumococcal carriage in children has been extensively studied, but carriage in healthy adults and its relationship to invasive pneumococcal disease (IPD) is less understood., Methods: Nasal wash samples from adults without close contact with young children (Liverpool, UK), 2011-2019, were cultured, and culture-negative samples tested by PCR. Pneumococcal carriage in adults 18-44 years was compared with carriage among PCV-vaccinated children 13-48 months (nasopharyngeal swabs, Thames Valley, UK) and IPD data for England for the same ages for 2014-2019. Age-group specific serotype invasiveness was calculated and used with national IPD data to estimate carriage serotype distributions for adults aged 65+ years., Results: In total 98 isolates (97 carriers) were identified from 1,631 adults aged 18+ years (age and sex standardized carriage prevalence 6.4%), with only three identified solely by PCR. Despite different carriage and IPD serotype distributions between adults and children, serotype invasiveness was highly correlated (R=0.9). Serotypes 3, 37 and 8 represented a higher proportion of adult carriage than expected from direct low-level transmission from children to adults. The predicted carriage serotype distributions for 65+ years aligned more closely with the carriage serotype distribution for young adults than young children., Conclusions: The nasal wash technique is highly sensitive; additional benefit of PCR is limited. Comparison of carriage serotype distributions suggests some serotypes may be circulating preferentially within these specific young adults. Our data suggest that for some serotypes carried by adults 65+ years, other adults may be an important reservoir for transmission. Age groups such as older children should also be considered., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: In Malawi, the national pneumococcal conjugate vaccine (PCV13) demonstrated less herd immunity than the USA, likely due to higher natural pneumococcal carriage rates. We assessed PCV13 efficacy against experimental pneumococcal carriage in healthy Malawian adults. We explored how natural carriage (pneumococcal carriage of any other serotype apart from 6B) influenced experimental carriage rates and vaccine efficacy., Methods: Healthy adults aged 18-40 were randomly assigned PCV13 (n=98) or saline (n=106), followed by intranasal SPN 6B inoculation at 20,000 (n=40), 80,000 (n=74), or 160,000 (n=90) CFU/100µl, 28 days post-vaccination. We evaluated natural and experimental pneumococcal carriage before and after vaccination on days 2, 7, and 14 post-inoculation using culture and multiplex qPCR targeting lytA/cpsA genes and compared carriage rates by vaccination status., Results: Of 204 participants, 19.6% (40) exhibited experimental carriage, detected by culture and 25.5% (52) by qPCR. Vaccinated individuals had lower experimental carriage rates (10.2%, n=10/98) compared to the placebo group (28.3%, n=30/106). This difference in vaccine efficacy was more pronounced in participants without natural carriage (PCV13=8% n=6/75 vs. placebo=25.9%, n=21/81) compared to those with natural carriage (PCV13=14.8%, n=4/27 vs. placebo=26.5%, n=9/34). Using a log-binomial model, vaccine effectiveness (VE) was 62%, whether assessed by culture or qPCR. Natural carriers had a lower VE of 52% compared to participants with no natural carriage (VE=69%)., Conclusion: We have shown that PCV13 VE estimate (62%) is robust whether carriage is assessed by culture or qPCR. PCV13 had lower VE in natural carriers compared to those without natural carriage at the inoculation visit., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Tuberculosis (TB) caused 1.5 million deaths in 2020, making it the leading infectious killer after COVID-19. Bacille Calmette-Guerin (BCG) is the only licensed vaccine against TB but has sub-optimal efficacy against pulmonary TB and reduced effectiveness in regions close to the equator with high burden. Efforts to find novel vaccines are hampered due to the need for large-scale, prolonged, and costly clinical trials. Controlled human infection models (CHIMs) for TB may be used to accelerate vaccine development by ensuring only the most promising vaccine candidates are selected for phase 3 trials, but it is not currently possible to give participants Mycobacterium tuberculosis as a challenge agent. This study aims to replicate and refine an established BCG CHIM at the Liverpool School of Tropical Medicine. Participants will receive an intradermal injection with licensed BCG vaccine (Statens Serum Institut strain). In phase A, participants will undergo punch biopsy two weeks after administration, paired with minimally invasive methods of skin sampling (skin swab, microbiopsy, skin scrape). BCG detection by classical culture and molecular methods will be compared between these techniques and gold standard punch biopsy. Techniques meeting our pre-defined sensitivity and specificity criteria will be applied in Phase B to longitudinally assess intradermal BCG growth two, seven and fourteen days after administration. We will also measure compartmental immune responses in skin, blood and respiratory mucosa in Phase B. This feasibility study will transfer and refine an existing and safe model of BCG controlled human infection. Longitudinal BCG quantification has the potential to increase model sensitivity to detect vaccine and therapeutic responses. If successful, we aim to transfer the model to Malawi in future studies, a setting with endemic TB disease, to accelerate development of vaccines and therapeutics relevant for underserved populations who stand to benefit the most. Registration: ISRCTN: ISRCTN94098600 and ClinicalTrials.gov: NCT05820594., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Carter E et al.)

Background: Pneumococcal carriage is the primary reservoir for transmissionand a prerequisite for invasive pneumococcal disease. Pneumococcal Conjugate Vaccine 13 (PCV13) showed a 62% efficacy in protection against experimental Streptococcus pneumoniae serotype 6B (Spn6B) carriage in a controlled human infection model (CHIM) of healthy Malawian adults. We, therefore, measured humoral responses to experimental challenge and PCV-13 vaccination and determined the association with protection against pneumococcal carriage., Methods: We vaccinated 204 young, healthy Malawian adults with PCV13 or placebo and nasally inoculated them with Spn6B at least four weeks post-vaccination to establish carriage. We collected peripheral blood and nasal lining fluid at baseline, 4 weeks post-vaccination (7 days pre-inoculation), 2, 7, 14 and > 1 year post-inoculation. We measured the concentration of anti-serotype 6B Capsular Polysaccharide (CPS) Immunoglobulin G (IgG) and IgA antibodies in serum and nasal lining fluid using the World Health Organization (WHO) standardised enzyme-linked immunosorbent assay (ELISA)., Results: PCV13-vaccinated adults had higher serum IgG and nasal IgG/IgA anti-Spn6B CPS-specific binding antibodies than placebo recipients 4 to 6 weeks post-vaccination, which persisted for at least a year after vaccination. Nasal challenge with Spn6B did not significantly alter serum or nasal anti-CPS IgG binding antibody titers with or without experimental pneumococcal carriage. Pre-challenge titers of PCV13-induced serum IgG and nasal IgG/IgA anti-Spn6B CPS binding antibodies did not significantly differ between those that got experimentally colonised by Spn6B compared to those that did not., Conclusion: This study demonstrates that despite high PCV13 efficacy against experimental Spn6B carriage in young, healthy Malawian adults, robust vaccine-induced systemic and mucosal anti-Spn6B CPS binding antibodies did not directly relate to protection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier India Pvt Ltd. All rights reserved.)

Background: Hospital admission due to breathlessness carries a significant burden to patients and healthcare systems, particularly impacting people in low-income countries. Prompt appropriate treatment is vital to improve outcomes, but this relies on accurate diagnostic tests which are of limited availability in resource-constrained settings. We will provide an accurate description of acute breathlessness presentations in a multicentre prospective cohort study in Malawi, a low resource setting in Southern Africa, and explore approaches to strengthen diagnostic capacity., Objectives: Primary objective: Delineate between causes of breathlessness among adults admitted to hospital in Malawi and report disease prevalence. Secondary objectives : Determine patient outcomes, including mortality and hospital readmission 90 days after admission; determine the diagnostic accuracy of biomarkers to differentiate between heart failure and respiratory infections (such as pneumonia) including brain natriuretic peptides, procalcitonin and C-reactive protein., Methods: This is a prospective longitudinal cohort study of adults (≥18 years) admitted to hospital with breathlessness across two hospitals: 1) Queen Elizabeth Central Hospital, Blantyre, Malawi; 2) Chiradzulu District Hospital, Chiradzulu, Malawi. Patients will be consecutively recruited within 24 hours of emergency presentation and followed-up until 90 days from hospital admission. We will conduct enhanced diagnostic tests with robust quality assurance and quality control to determine estimates of disease pathology. Diagnostic case definitions were selected following a systematic literature search., Discussion: This study will provide detailed epidemiological description of adult hospital admissions due to breathlessness in low-income settings, which is currently poorly understood. We will delineate between causes using established case definitions and conduct nested diagnostic evaluation. The results have the potential to facilitate development of interventions targeted to strengthen diagnostic capacity, enable prompt and appropriate treatment, and ultimately improve both patient care and outcomes., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Spencer SA et al.)

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Doherty K et al.)