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Background: Efforts to control tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis (Mtb), have been hampered by the immense variability in protection from BCG vaccination. While BCG protects young children from some forms of TB disease, long-term protection against pulmonary disease is more limited, suggesting a poor memory response. New vaccines or vaccination strategies are required to have a realistic chance of eliminating TB disease. In TB endemic areas, routine immunization occurs during the neonatal period and as such, we hypothesized that inadequate protective immunity elicited by BCG vaccination could be the result of the unique early-life immune landscape. Interleukin (IL)-27 is a heterodimeric cytokine with immune suppressive activity that is elevated in the neonatal period., Objective: We investigated the impact of IL-27 on regulation of immune responses during neonatal BCG vaccination and protection against Mtb., Methods: Here, we used a novel model of neonatal vaccination and adult aerosol challenge that models the human timeline of vaccine delivery and disease transmission., Results: Overall, we observed improved control of Mtb in mice unresponsive to IL-27 (IL-27Rα -/- ) that was consistent with altered expression patterns of IFN-γ and IL-17 in the lungs. The balance of these cytokines with TNF-α expression may be key to effective bacterial clearance., Conclusions: Our findings suggest the importance of evaluating new vaccines and approaches to combat TB in the neonatal population most likely to receive them as part of global vaccination campaigns. They further indicate that temporal strategies to antagonize IL-27 during early life vaccination may improve protection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Bradford, Ryan, Divens, Povroznik, Bonigala and Robinson.)

Bacille Calmette Guérin (BCG) is a live-attenuated vaccine for protection against Mycobacterium tuberculosis . Despite high disease protection in infancy and early childhood, it generates poor long-term protection against pulmonary tuberculosis. We hypothesized that the unique immune profile that includes elevated interleukin (IL)-27, contributes to insufficient protection from routine neonatal BCG administration. Using a novel method to obtain neonatal progenitors, we showed that neonatal bone marrow-derived dendritic cells (BMDCs) increase production of IL-27 following BCG stimulation. To study the effect of IL-27 on BMDCs, we utilized mice deficient for IL-27 receptor-α (KO). We observed greater BCG clearance and elevated IL-12 production in the neonatal KO BMDCs compared to WT. BMDCs from KO neonates in turn stimulated more interferon-γ production from CD4 + T cells isolated from BCG-vaccinated mice than WT counterparts. To further confirm the importance of these findings, C57BL/6 mice were vaccinated as neonates in line with the approach to human vaccination in high TB burden regions. IL-27 levels progressively increased through 5 weeks and were significantly elevated in mice vaccinated with BCG compared to controls. The impact of IL-27 production on clearance of BCG was significant as KO mice cleared BCG from peripheral tissues that persisted in WT mice 5 weeks post-vaccination. These results are the first to highlight the suppressive role of IL-27 on DCs in the neonatal period and the impact on neonatal immune responses to BCG., Competing Interests: 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., (© 2022 The Author(s).)

We perform electron diffraction of 1,4-dichlorobenzene (C 6 H 4 Cl 2 , referred to as 2ClB) embedded in superfluid helium droplets to investigate the structure evolution of cluster growth. Multivariable linear regression fittings are used to determine the concentration and the best model structures of the clusters. At a droplet source temperature of 22 K with droplets containing on average 5000 He atoms, the fitting results agree with the doping statistics modeled using the Poisson distribution: the largest molecular clusters are tetramers, while the abundances of monomers and dimers are the highest and are similar. Molecular dimers of 2ClB are determined to have a parallel structure with a 60° rotation for the Cl-Cl molecular axes. However, a better agreement between experiment and fitting is obtained by reducing the interlayer distance that had been calculated using the density functional theory for dimers. Further calculations using the highest level quantum mechanical calculations prove that the reduction in interlayer distance does not significantly increase the energy of the dimer. Cluster trimers adopt a dimer structure with the additional monomer slanted against the dimer, and tetramers take on a stacked structure. The structure evolution with cluster size is extraordinary, because from trimer to tetramer, one monomer needs to be rearranged, and neither the trimer nor the tetramer adopts the corresponding global minimum structure obtained using high level coupled-cluster theory calculations. This phenomenon may be related to the fast cooling process in superfluid helium droplets during cluster formation.

We report the electron diffraction of cationic pyrene (C 16 H 10 ) clusters embedded in superfluid helium droplets. The diffraction profile contains a significant contribution from helium, but interferences of atomic pairs of pyrene are still recognizable. From least-squares fittings, we determine an interlayer distance of 3.0 Å for the cationic cluster, shortened from 3.5 Å in neutral clusters. The relative contributions of dimers and trimers are about 2:1, in qualitative agreement with the doping statistics. Limited by the detection range of the experimental data, we cannot distinguish further structure details. The predominant contribution of helium also prevents observations of the solvation shell of the ionic cluster. Nevertheless, the success of this experiment demonstrates the feasibility of electron diffraction from an ionic all-light-atom system, dispelling the concern over limited particle concentration of ionic species in the diffraction region, and the need of heavy atoms for diffraction intensity.

We report electron diffraction of cationic argon nanoclusters embedded in superfluid helium droplets. Superfluid helium droplets are first doped with neutral argon atoms to form nanoclusters, and then the doped droplets are ionized by electrons. The much lower ionization energy of argon ensures that the positive charge resides on the Ar nanocluster. Using different stagnation temperatures and therefore droplets with different sizes, we have been able to preferentially form a small ionic cluster containing 2-4 Ar atoms and a larger cluster containing 7-11 atoms. The fitting results of the diffraction profiles agree with structures reported from theoretical calculations, containing a cationic trimer core with the remaining atoms largely neutral. This work testifies to the feasibility of performing electron diffraction from ionic species embedded in superfluid helium droplets, dispelling the concern over the particle density in the diffraction region. However, the large number of neutral helium atoms surrounding the cationic nanoclusters poses a challenge for the detection of the helium solvation layer, and the detection of which awaits further technological improvements.

Whole cell vaccines are frequently the first generation of vaccines tested for pathogens and can inform the design of subsequent acellular or subunit vaccines. For respiratory pathogens, administration of vaccines at the mucosal surface can facilitate the generation of a localized mucosal immune response. Here, we examined the innate and vaccine-induced immune responses to infection by two respiratory pathogens: Bordetella pertussis and Pseudomonas aeruginosa . In a model of intranasal administration of whole cell vaccines (WCVs) with the adjuvant curdlan, we examined local and systemic immune responses following infection. These studies showed that intranasal vaccination with a WCV led to a reduction of the bacterial burden in the airways of animals infected with the respective pathogen. However, there were unique changes in the cytokines produced, cells recruited, and inflammation at the site of infection. Both mucosal vaccinations induced antibodies that bind the target pathogen, but linear regression and principal component analysis revealed that protection from these pathogens is not solely related to antibody titer. Protection from P. aeruginosa correlated to a reduction in lung weight, blood lymphocytes and neutrophils, and the cytokines IL-6, TNF-α, KC/GRO, and IL-10, and promotion of serum IgG antibodies and the cytokine IFN-γ in the lung. Protection from B. pertussis infection correlated strongly with increased anti- B-pertussis serum IgG antibodies. These findings reveal valuable correlates of protection for mucosal vaccination that can be used for further development of both B. pertussis and P. aeruginosa vaccines.

We report experimental results from electron diffraction of CS 2 nanoclusters embedded in superfluid helium droplets. From detailed measurements of the sizes of doped droplets, we can model the doping statistics under different experimental conditions, thereby obtaining the range of cluster sizes of CS 2 . Using a least squares fitting procedure, we can then determine the structures and contributions of dimers, trimers, and tetramers embedded in small droplets. While dimers prefer a stable gas phase structure, trimers and tetramers seem to forgo the highly symmetric gas phase structures and prefer compact cuts from the crystalline structure of CS 2 . In larger droplets containing more than 12 CS 2 monomers, the diffraction profile is consistent with a three-dimensional nanostructure of bulk CS 2 . This work demonstrates the feasibility of electron diffraction for in situ monitoring of nanocluster formation in superfluid helium droplets.

Neonates are at increased risk for bacterial sepsis. We established that the immune-suppressive cytokine interleukin-27 (IL-27) is elevated in neonatal mice. Similarly, human cord blood-derived macrophages express IL-27 genes and secrete more cytokine than macrophages from adults. In the present work, we hypothesized that increased levels of IL-27 predispose neonatal mice to more severe infection during Gram-negative sepsis. Serum IL-27 levels continued to rise during infection. Peripheral tissue analysis revealed systemic IL-27 expression, while myeloid cell profiling identified Gr-1- and F4/80-expressing cells as the most abundant producers of IL-27 during infection. Increased IL-27 levels were consistent with increased mortality that was improved in IL-27 receptor α (IL-27Rα) -/- mice that lack a functional IL-27 receptor. Infected IL-27Rα -/- pups also exhibited improved weight gain and reduced morbidity. This was consistent with reduced bacterial burdens and more efficient bacterial killing by Ly6B.2 + myeloid cells and macrophages compared to WT neonates. Live animal imaging further supported a more severe and disseminated infection in WT neonates. This is the first report to describe the impact of elevated early-life IL-27 on the host response in a neonatal infection model while also defining the cell and tissue sources of cytokine. IL-27 is frequently associated with suppressed inflammation. In contrast, our findings demonstrate that IL-27 indirectly promotes an inflammatory cytokine response during neonatal sepsis by directly compromising control of bacteria that drive the inflammatory response. Collectively, our results suggest that IL-27 represents a therapeutic target to limit susceptibility and improve infectious outcomes in neonatal sepsis., (Copyright © 2020 Seman et al.)

We report suppression of multiphoton ionization (MPI) of aniline doped large superfluid helium droplets containing over 5 × 10 6 atoms. In contrast, surface-bound sodium atoms and dimers are readily desorbed and ionized. Adequacy of the experimental conditions is also confirmed from ejection of embedded aniline cations from smaller droplets containing multiple cations, and MPI of gaseous aniline. The photoelectrons have a mean-free-path of less than 1 nm and a thermalization distance of 10 nm. In a droplet with a diameter of over 70 nm, effective charge recombination within the droplet is expected., Competing Interests: Declaration of interests 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.

Pseudomonas aeruginosa is an opportunistic pathogen causing acute and chronic respiratory infections associated with morbidity and mortality, especially in patients with cystic fibrosis. Vaccination against P. aeruginosa before colonization may be a solution against these infections and improve the quality of life of at-risk patients. To develop a vaccine against P. aeruginosa , we formulated a novel peptide-based P. aeruginosa subunit vaccine based on the extracellular regions of one of its major siderophore receptors, FpvA. We evaluated the effectiveness and immunogenicity of the FpvA peptides conjugated to keyhole limpet hemocyanin (KLH) with the adjuvant curdlan in a murine vaccination and challenge model. Immunization with the FpvA-KLH vaccine decreased the bacterial burden and lung edema after P. aeruginosa challenge. Vaccination with FpvA-KLH lead to antigen-specific IgG and IgM antibodies in sera, and IgA antibodies in lung supernatant. FpvA-KLH immunized mice had an increase in recruitment of CD11b + dendritic cells as well as resident memory CD4 + T cells in the lungs compared to non-vaccinated challenged mice. Splenocytes isolated from vaccinated animals showed that the FpvA-KLH vaccine with the adjuvant curdlan induces antigen-specific IL-17 production and leads to a Th17 type of immune response. These results indicate that the intranasal FpvA-KLH conjugate vaccine can elicit both mucosal and systemic immune responses. These observations suggest that the intranasal peptide-based FpvA-KLH conjugate vaccine with curdlan is a potential vaccine candidate against P. aeruginosa pneumonia., (Copyright © 2019 Sen-Kilic, Blackwood, Boehm, Witt, Malkowski, Bevere, Wong, Hall, Bradford, Varney, Damron and Barbier.)

Current acellular pertussis vaccines fall short of optimal protection against the human respiratory pathogen Bordetella pertussis resulting in increased incidence of a previously controlled vaccine- preventable disease. Natural infection is known to induce a protective mucosal immunity. Therefore, in this study, we aimed to use acellular pertussis vaccines to recapitulate these mucosal immune responses. We utilized a murine immunization and challenge model to characterize the efficacy of intranasal immunization (IN) with DTaP vaccine or DTaP vaccine supplemented with curdlan, a known Th1/Th17 promoting adjuvant. Protection from IN delivered DTaP was compared to protection mediated by intraperitoneal injection of DTaP and whole-cell pertussis vaccines. We tracked fluorescently labeled DTaP after immunization and detected that DTaP localized preferentially in the lungs while DTaP with curdlan was predominantly in the nasal turbinates. IN immunization with DTaP, with or without curdlan adjuvant, resulted in anti- B. pertussis and anti-pertussis toxin IgG titers at the same level as intraperitoneally administered DTaP. IN immunization was able to protect against B. pertussis challenge and we observed decreased pulmonary pro-inflammatory cytokines, neutrophil infiltrates in the lung, and bacterial burden in the upper and lower respiratory tract at day 3 post challenge. Furthermore, IN immunization with DTaP triggered mucosal immune responses such as production of B. pertussis -specific IgA, and increased IL-17A. Together, the induction of a mucosal immune response and humoral antibody-mediated protection associated with an IN administered DTaP and curdlan adjuvant warrant further exploration as a pertussis vaccine candidate formulation., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2019.)

Microbial infections early in life remain a major cause of infant mortality worldwide. This is consistent with immune deficiencies in this population. Interleukin (IL)-27 is suppressive toward a variety of immune cell types, and we have shown that the production of IL-27 is elevated in humans and mice early in life. We hypothesize that elevated levels of IL-27 oppose protective responses to infection during the neonatal period. In this study, we extended previous findings in neonatal mice to identify a population of IL-27 producers that express Gr-1 and were further identified as myeloid-derived suppressor cells (MDSCs) based on the expression of surface markers and functional studies. In neonates, MDSCs are more abundant and contribute to the elevated pool of IL-27 in this population. Although the ability of MDSCs to regulate T lymphocyte activation has been well-studied, sparingly few studies have investigated the influence of MDSCs on innate immune function during bacterial infection. We demonstrate that macrophages are impaired in their ability to control growth of Escherichia coli when cocultured with MDSCs. This bacterium is a significant concern for neonates as a common cause of bacterial sepsis and meningitis. The suppressive effect of MDSCs on macrophage function is mediated by IL-27; inclusion of a reagent to neutralize IL-27 promotes improved control of bacterial growth. Taken together, these results suggest that the increased abundance of MDSCs may contribute to early life susceptibility to infection and further highlight production of IL-27 as a novel MDSC mechanism to suppress immunity., (© 2018 Australasian Society for Immunology Inc.)

Hematopoietic stem and progenitor cell (HSPC) compartments are altered to direct immune responses to infection. Their roles during immunization are not well-described. To elucidate mechanisms for waning immunity following immunization with acellular vaccines (ACVs) against Bordetella pertussis ( Bp ), we tested the hypothesis that immunization with Bp ACVs and whole cell vaccines (WCVs) differ in directing the HSPC characteristics and immune cell development patterns that ultimately contribute to the types and quantities of cells produced to fight infection. Our data demonstrate that compared to control and ACV-immunized CD-1 mice, immunization with an efficacious WCV drives expansion of hematopoietic multipotent progenitor cells (MPPs), increases circulating white blood cells (WBCs), and alters the size and composition of lymphoid organs. In addition to MPPs, common lymphoid progenitor (CLP) proportions increase in the bone marrow of WCV-immunized mice, while B220 + cell proportions decrease. Upon subsequent infection, increases in maturing B cell populations are striking in WCV-immunized mice. RNAseq analyses of HSPCs revealed that WCV and ACV-immunized mice vastly differ in developing VDJ gene segment diversity. Moreover, gene set enrichment analyses demonstrate WCV-immunized mice exhibit unique gene signatures that suggest roles for interferon (IFN) induced gene expression. Also observed in naïve infection, these IFN stimulated gene (ISG) signatures point toward roles in cell survival, cell cycle, autophagy, and antigen processing and presentation. Taken together, these findings underscore the impact of vaccine antigen and adjuvant content on skewing and/or priming HSPC populations for immune response.

Bordetella pertussis is the primary causative agent of pertussis (whooping cough), which is a respiratory infection that leads to a violent cough and can be fatal in infants. There is a need to develop more effective vaccines because of the resurgence of cases of pertussis in the United States since the switch from the whole-cell pertussis vaccines (wP) to the acellular pertussis vaccines (aP; diphtheria-tetanus-acellular-pertussis vaccine/tetanus-diphtheria-pertussis vaccine). Adenylate cyclase toxin (ACT) is a major virulence factor of B. pertussis that is (i) required for establishment of infection, (ii) an effective immunogen, and (iii) a protective antigen. The C-terminal repeats-in-toxin domain (RTX) of ACT is sufficient to induce production of toxin-neutralizing antibodies. In this study, we characterized the effectiveness of vaccines containing the RTX antigen against experimental murine infection with B. pertussis RTX was not protective as a single-antigen vaccine against B. pertussis challenge, and adding RTX to 1/5 human dose of aP did not enhance protection. Since the doses of aP used in murine studies are not proportionate to mouse/human body masses, we titrated the aP from 1/20 to 1/160 of the human dose. Mice receiving 1/80 human aP dose had bacterial burden comparable to those of naive controls. Adding RTX antigen to the 1/80 aP base resulted in enhanced bacterial clearance. Inclusion of RTX induced production of antibodies recognizing RTX, enhanced production of anti-pertussis toxin, decreased secretion of proinflammatory cytokines, such as interleukin-6, and decreased recruitment of total macrophages in the lung. This study shows that adding RTX antigen to an appropriate dose of aP can enhance protection against B. pertussis challenge in mice., (Copyright © 2018 American Society for Microbiology.)