Formulation of a mmaA4 Gene Deletion Mutant of Mycobacterium bovis BCG in Cationic Liposomes Significantly Enhances Protection against Tuberculosis

A new vaccination strategy is urgently needed for improved control of the global tuberculosis (TB) epidemic. Using a mouse aerosol Mycobacterium tuberculosis challenge model, we investigated the protective efficacy of a mmaA4 gene deletion mutant of Mycobacterium bovis BCG (ΔmmaA4BCG) formulated in dimethyl dioctadecyl ammonium bromide (DDA) – D(+) trehalose 6,6 dibenenate (TDB) (DDA/TDB) adjuvant. In previous studies, deletion of the mmaA4 gene was shown to reduce the suppression of IL-12 production often seen after mycobacterial infections. While the non-adjuvanted ΔmmaA4BCG strain did not protect mice substantially better than conventional BCG against a tuberculous challenge in four protection experiments, the protective responses induced by the ΔmmaA4BCG vaccine formulated in DDA/TDB adjuvant was consistently increased relative to nonadjuvanted BCG controls. Furthermore, the ΔmmaA4BCG-DDA/TDB vaccine induced significantly higher frequencies of multifunctional (MFT) CD4 T cells expressing both IFNγ and TNFα (double positive) or IFNγ, TNFα and IL-2 (triple positive) than CD4 T cells derived from mice vaccinated with BCG. These MFT cells were characterized by having higher IFNγ and TNFα median fluorescence intensity (MFI) values than monofunctional CD4 T cells. Interestingly, both BCG/adjuvant and ΔmmaA4BCG/adjuvant formulations induced significantly higher frequencies of CD4 T cells expressing TNFα and IL-2 than nonadjuvanted BCG or ΔmmaA4BCG vaccines indicating that BCG/adjuvant mixtures may be more effective at inducing central memory T cells. Importantly, when either conventional BCG or the mutant were formulated in adjuvant and administered to SCID mice or immunocompromised mice depleted of IFNγ, significantly lower vaccine-derived mycobacterial CFU were detected relative to immunodeficient mice injected with non-adjuvanted BCG. Overall, these data suggest that immunization with the ΔmmaA4BCG/adjuvant formulation may be an effective, safe, and relatively inexpensive alternative to vaccination with conventional BCG

H5N1 Whole-Virus Vaccine Induces Neutralizing Antibodies in Humans Which Are Protective in a Mouse Passive Transfer Model

BACKGROUND: Vero cell culture-derived whole-virus H5N1 vaccines have been extensively tested in clinical trials and consistently demonstrated to be safe and immunogenic; however, clinical efficacy is difficult to evaluate in the absence of wide-spread human disease. A lethal mouse model has been utilized which allows investigation of the protective efficacy of active vaccination or passive transfer of vaccine induced sera following lethal H5N1 challenge. METHODS: We used passive transfer of immune sera to investigate antibody-mediated protection elicited by a Vero cell-derived, non-adjuvanted inactivated whole-virus H5N1 vaccine. Mice were injected intravenously with H5N1 vaccine-induced rodent or human immune sera and subsequently challenged with a lethal dose of wild-type H5N1 virus. RESULTS: Passive transfer of H5N1 vaccine-induced mouse, guinea pig and human immune sera provided dose-dependent protection of recipient mice against lethal challenge with wild-type H5N1 virus. Protective dose fifty values for serum H5N1 neutralizing antibody titers were calculated to be ≤1∶11 for all immune sera, independently of source species. CONCLUSIONS: These data underpin the confidence that the Vero cell culture-derived, whole-virus H5N1 vaccine will be effective in a pandemic situation and support the use of neutralizing serum antibody titers as a correlate of protection for H5N1 vaccines

Respiratory and immune response to maximal physical exertion following exposure to secondhand smoke in healthy adults

© 2012 The Authors. Published by PLOS. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.1371/journal.pone.0031880We assessed the cardiorespiratory and immune response to physical exertion following secondhand smoke (SHS) exposure through a randomized crossover experiment. Data were obtained from 16 (8 women) non-smoking adults during and following a maximal oxygen uptake cycling protocol administered at baseline and at 0-, 1-, and 3- hours following 1-hour of SHS set at bar/restaurant carbon monoxide levels. We found that SHS was associated with a 12% decrease in maximum power output, an 8.2% reduction in maximal oxygen consumption, a 6% increase in perceived exertion, and a 6.7% decrease in time to exhaustion (P<0.05). Moreover, at 0-hours almost all respiratory and immune variables measured were adversely affected (P<0.05). For instance, FEV 1 values at 0-hours dropped by 17.4%, while TNF-α increased by 90.1% (P<0.05). At 3-hours mean values of cotinine, perceived exertion and recovery systolic blood pressure in both sexes, IL4, TNF-α and IFN-γ in men, as well as FEV 1/FVC, percent predicted FEV 1, respiratory rate, and tidal volume in women remained different compared to baseline (P<0.05). It is concluded that a 1-hour of SHS at bar/restaurant levels adversely affects the cardiorespiratory and immune response to maximal physical exertion in healthy nonsmokers for at least three hours following SHS. © 2012 Flouris et al.Published versio

Delivery of GM-CSF to Protect against Influenza Pneumonia.

Since adaptive immunity is thought to be central to immunity against influenza A virus (IAV) pneumonias, preventive strategies have focused primarily on vaccines. However, vaccine efficacy has been variable, in part because of antigenic shift and drift in circulating influenza viruses. Recent studies have highlighted the importance of innate immunity in protecting against influenza.Granulocyte-macrophage colony stimulating factor (GM-CSF) contributes to maturation of mononuclear phagocytes, enhancing their capacity for phagocytosis and cytokine production.Overexpression of granulocyte macrophage-colony stimulating factor (GM-CSF) in the lung of transgenic mice provides remarkable protection against IAV, which depends on alveolar macrophages (AM). In this study, we report that pulmonary delivery of GM-CSF to wild type young and aged mice abrogated mortality from IAV.We also demonstrate that protection is species specific and human GM-CSF do not protect the mice nor stimulates mouse immunity. We also show that IAV-induced lung injury is the culprit for side-effects of GM-CSF in treating mice after IAV infection, and introduce a novel strategy to deliver the GM-CSF to and retain it in the alveolar space even after IAV infection

Boosting efferocytosis in alveolar space using BCG vaccine to protect host against influenza pneumonia

<div><p>Efferocytosis by alveolar phagocytes (APs) is pivotal in maintenance of lung homeostasis. Increased efferocytosis by APs results in protection against lethal acute lung injury due to pulmonary infections whereas defective efferocytosis by APs results in chronic lung inflammation. In this report, we show that pulmonary delivery of Bacillus Calmette-Guerin (BCG) significantly enhances efferocytosis by APs. Increased efferocytosis by APs maintains lung homeostasis and protects mice against lethal influenza pneumonia. Intranasally treated wild type C57Bl/6 (WT) mice with BCG showed significant increase in APs efferocytosis in vivo compared to their PBS-treated counterparts. All BCG-treated WT mice survived lethal influenza A virus (IAV) infection whereas all PBS-treated mice succumbed. BCG-induced resistance was abrogated by depleting AP prior to IAV infection. BCG treatment increased uptake, and digestion/removal of apoptotic cells by APs. BCG significantly increased the expression of TIM4 on APs and increased expression of Rab5 and Rab7. We demonstrated that increased efferocytosis by APs through pulmonary delivery of BCG initiated rapid clearance of apoptotic cells from the alveolar space, maintained lung homeostasis, reduced inflammation and protected host against lethal IAV pneumonia.</p></div

Deletion of FoxN1 in the Thymic Medullary Epithelium Reduces Peripheral T Cell Responses to Infection and Mimics Changes of Aging

Aging increases susceptibility to infection, in part because thymic involution culminates in reduced naïve T-lymphocyte output. Thymic epithelial cells (TECs) are critical to ensure normal maturation of thymocytes and production of peripheral T cells. The forkhead-class transcription factor, encoded by FoxN1, regulates development, differentiation, and function of TECs, both in the prenatal and postnatal thymus. We recently showed that expression of FoxN1, by keratin 14 (K14)expressing epithelial cells is essential for maintenance of thymic medullary architecture, and deletion of FoxN1 in K14 promoter-driven TECs inhibited development of mature TECs and reduced the number of total thymocytes. These findings are reminiscent of changes observed during normal thymic aging. In the current report, we compared the effects of K14driven FoxN1 deletion on peripheral T cell function in response to influenza virus infection with those associated with normal aging in a mouse model. FoxN1-deleted mice had reduced numbers of peripheral CD62L+CD442 naïve T-cells. In addition, during influenza infection, these animals had reduced antigen-specific CD8+ T-cell and IgG responses to influenza virus, combined with increased lung injury, weight loss and mortality. These findings paralleled those observed in aged wild type mice, providing the first evidence that K14-mediated FoxN1 deletion causes changes in T-cell function that mimic thos

BCG vaccination increases Tim4 expression in APs.

<p><b>(</b>A) Expression of Tim-4 was determined in BCG vaccinated and control PBS treated mice before and after influenza infection using western blotting. WT mice were treated with either BCG or PBS and infected with lethal PR8 as described in the legend of Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180143#pone.0180143.g001" target="_blank">1D</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180143#pone.0180143.g002" target="_blank">2</a>. Two days after influenza infection, BAL cells were collected, pooled and lysed as delineated in materials and methods. Each lane is pool of BALs from 5 mice. Equal amount of protein extracts from each group were loaded into each lane. (B) Densitometry analysis of band-intensity using Image Lab software (Molecular Imager Gel Doc system from Bio-Rad) is shown. Data is representation of band-intensities of 3 independent blots. Error bars show SEM. DPI: Days Post Infection.</p

Delivery of BCG to alveolar space enhances efferocytosis by APs.

<p>Staurosporine-induced apoptotic mouse lung epithelial (MLE) cells were labeled with CFSE and intranasally transferred to mice treated intranasally with BCG or PBS. Bronchoalveolar lavage (BAL) cells were collected 1 h later and stained with F4/80. (A) Flow cytometry analysis of individual mice is shown. (B) Average of free CFSE+apoptotic MLE cells and efferocytosis efficiency from panel A. Efferocytosis efficiency was calculated as percent of efferocytosed CFSE+ apoptotic cells (CFSE+ and F4/80+F4/80+double-positive cells)/total percent of CSFE^+ve cells. A representative of two to four independent experiments with 3–5 mice per group are shown. Error bars show SEM. (C<b>) Localization of apoptotic cells insideAPs.</b> WT mice were treated as in panel A. Apoptotic MLE cells were stained with red pHrodo® and administered intranasally to PBS-treated (upper row) control and BCG-treated (lower row). BAL cells were collected and subjected to confocal microscopy.60x Magnification. A representative of two independent experiments with 3–4 mice per group is depicted. (D-F<b>) Pulmonary treatment with BCG rapidly and efficiently boosts efferocytosis by APs in disease condition.</b> WT mice were treated intranasally with BCG or PBS. Two days later, all mice were infected with PR8 strain of influenza virus. Two days after IAV infection, all mice received CFSE-labeled apoptotic MLE cells intranasally and the apoptotic cell clearance was measured by flow cytometry, as described above. (D) Flow cytometry analysis of each individual mouse either treated with PBS (top row) or BCG (bottom row) is shown. (E)Average percent of free CFSE+ apoptotic MLE cells and efferocytosis efficiency from panel (D) are shown. Efferocytosis efficiency of APs was calculated using the formula from panel B. A representative of three to four independent experiments shown with 3–5 mice per group. Error bars show SEM. (F<b>) Visualization of the engulfed apoptotic cells inside the APs in BCG vaccinated and control mice after IAV infection.</b> WT mice were treated as in panel D. Two days after IAV infection, all mice received pHrodo®-labeled apoptotic MLE cells intranasally and the fate of apoptotic cells inside the AMs of PBS-treated (upper row) control and BCG-treated (lower row) mice was studied using confocal microscopy, as described for panel1C. 60x magnification. A representative of two independent experiments with 3 mice per group is depicted.</p