The currently available commercial human anthrax vaccine requires multiple injections for efficacy and has side effects due to its alum adjuvant. These factors limit its utility when immunizing ...exposed populations in emergent situations. We evaluated a novel mucosal adjuvant that consists of a nontoxic, water-in-oil nanoemulsion (NE). This material does not contain a proinflammatory component but penetrates mucosal surfaces to load antigens into dendritic cells. Mice and guinea pigs were intranasally immunized with recombinant Bacillus anthracis protective antigen (rPA) mixed in NE as an adjuvant. rPA-NE immunization was effective in inducing both serum anti-PA immunoglobulin G (IgG) and bronchial anti-PA IgA and IgG antibodies after either one or two mucosal administrations. Serum anti-PA IgG2a and IgG2b antibodies and PA-specific cytokine induction after immunization indicate a Th1-polarized immune response. rPA-NE immunization also produced high titers of lethal-toxin-neutralizing serum antibodies in both mice and guinea pigs. Guinea pigs nasally immunized with rPA-NE vaccine were protected against an intradermal challenge with ~1,000 times the 50% lethal dose (~1,000x LD₅₀) of B. anthracis Ames strain spores (1.38 x 10³ spores), which killed control animals within 96 h. Nasal immunization also resulted in 70% and 40% survival rates against intranasal challenge with 10x LD₅₀ and 100x LD₅₀ (1.2 x 10⁶ and 1.2 x 10⁷) Ames strain spores. Our results indicate that NE can effectively adjuvant rPA for intranasal immunization. This potentially could lead to a needle-free anthrax vaccine requiring fewer doses and having fewer side effects than the currently available human vaccine.
Nipah virus (NiV) is a paramyxovirus (genus Henipavirus) that can cause severe respiratory illness and encephalitis in humans. Transmission occurs through consumption of NiV-contaminated foods, and ...contact with NiV-infected animals or human body fluids. However, it is unclear whether aerosols derived from aforesaid sources or others also contribute to transmission, and current knowledge on NiV-induced pathogenicity after small-particle aerosol exposure is still limited.
Infectivity, pathogenicity, and real-time dissemination of aerosolized NiV in Syrian hamsters was evaluated using NiV-Malaysia (NiV-M) and/or its recombinant expressing firefly luciferase (rNiV-FlucNP).
Both viruses had an equivalent pathogenicity in hamsters, which developed respiratory and neurological symptoms of disease, similar to using intranasal route, with no direct correlations to the dose. We showed that virus replication was predominantly initiated in the lower respiratory tract and, although delayed, also intensely in the oronasal cavity and possibly the brain, with gradual increase of signal in these regions until at least day 5-6 postinfection.
Hamsters infected with small-particle aerosolized NiV undergo similar clinical manifestations of the disease as previously described using liquid inoculum, and exhibit histopathological lesions consistent with NiV patient reports. NiV droplets could therefore play a role in transmission by close contact.
The causative agent of anthrax, Bacillus anthracis, produces two toxins that contribute in part to its virulence. Lethal toxin is a metalloprotease that cleaves upstream mitogen-activated protein ...kinase kinases. Edema toxin is a calmodulin-dependent adenylate cyclase. Previous studies demonstrated that the anthrax toxins are important immunomodulators that promote immune evasion of the bacterium by suppressing activation of macrophages and dendritic cells. Here we showed that injection of sublethal doses of either lethal or edema toxin into mice directly inhibited the subsequent activation of T lymphocytes by T-cell receptor-mediated stimulation. Lymphocytes were isolated from toxin-injected mice after 1 or 4 days and stimulated with antibodies against CD3 and CD28. Treatment with either toxin inhibited the proliferation of T cells. Injection of lethal toxin also potently inhibited cytokine secretion by stimulated T cells. The effects of edema toxin on cytokine secretion were more complex and were dependent on the length of time between the injection of edema toxin and the isolation of lymphocytes. Treatment with lethal toxin blocked multiple kinase signaling pathways important for T-cell receptor-mediated activation of T cells. Phosphorylation of the extracellular signal-regulated kinase and the stress-activated kinase p38 was significantly decreased. In addition, phosphorylation of the serine/threonine kinase AKT and of glycogen synthase kinase 3 was inhibited in T cells from lethal toxin-injected mice. Thus, anthrax toxins directly act on T lymphocytes in a mouse model. These findings are important for future anthrax vaccine development and treatment.
Bacillus anthracis, the etiological agent of anthrax, is a gram-positive spore-forming bacterium. It produces edema toxin (EdTx), a powerful adenylate cyclase that increases cyclic AMP (cAMP) levels ...in host cells. Because other cAMP-increasing agents inhibit key macrophage (MΦ) functions, such as phagocytosis, it was hypothesized that EdTx would exhibit similar suppressive activities. Our previous GeneChip data showed that EdTx downregulated MΦ genes involved in actin cytoskeleton remodeling, including protein kinase A (PKA). To further examine the role of EdTx during anthrax pathogenesis, we explored the hypothesis that EdTx treatment leads to deregulation of the cAMP-dependent PKA system, resulting in impaired cytoskeletal functions essential for MΦ activity. Our data revealed that EdTx significantly suppressed human MΦ phagocytosis of Ames spores. Cytoskeletal changes, such as decreased cell spreading and lowered F-actin content, were also observed for toxin-treated MΦs. Further, EdTx altered the protein levels and activity of PKA and exchange protein activated by cAMP (Epac), a recently identified cAMP-binding molecule. By using PKA- and Epac-selective cAMP analogs, we confirmed the involvement of both pathways in the inhibition of MΦ functions elicited by EdTx-generated cAMP. These results suggested that EdTx weakened the host immune response by increasing cAMP levels, which then signaled via PKA and Epac to cripple MΦ phagocytosis and interfered with cytoskeletal remodeling.
•CT-2* induced higher levels of cytokine production (IL-12,IFN-g,TNFa).•CT-2* was down-regulated ex-vivo apoptosis of splenocytes.•CT-2* stimulated expression of the toll-like receptor (TLR-4) gene ...was much higher than CT did.•CT-2* modulated cytokine production and apoptosis in splenocytes of mice possibly through the TLR-4 signaling pathway.
Native cholera toxin (CT) and its mutated form (CT-2*) without ADP-ribosyltransferase activity differ in their immunomodulatory effects on host cells, and the mechanisms of these differences are poorly understood. In this study, we demonstrated that CT-2* induced higher levels of cytokine production and down-regulated ex-vivo apoptosis of splenocytes from C57BL/6 mice. After exposure of the splenocytes ex-vivo to CT or CT-2* (2μg/ml) for 48h, CT-2* stimulated expression of the toll-like receptor (TLR-4) gene was much higher and the cells produced increased levels of interleukin (IL)-12, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, compared to splenocytes of mice exposed to native CT. We confirmed these findings by observing that CT-2*, induced much lower levels of IL-12, IFN-γ, and TNF-α in a TLR-4 knockout macrophage cell line derived from C57BL/6 mice. In addition, while CT is known to stimulate apoptosis in splenocytes, we observed that CT-2* significantly down-regulated apoptosis (4.2%), compared to splenocytes exposed to CT (18.7%) or PBS (negative control, 8.5%). On the contrary, we noted both native CT and CT-2* to exhibit similar levels of apoptosis in TLR-4−/− cell line. Overall, the evidence supports the conclusion that CT-2* modulated cytokine production and apoptosis in splenocytes of mice possibly through the TLR-4 signaling pathway.
Melioidosis is an endemic disease caused by the bacterium Burkholderia pseudomallei. Concerns exist regarding B. pseudomallei use as a potential bio-threat agent causing persistent infections and ...typically manifesting as severe pneumonia capable of causing fatal bacteremia. Development of suitable therapeutics against melioidosis is complicated due to high degree of genetic and phenotypic variability among B. pseudomallei isolates and lack of data establishing commonly accepted strains for comparative studies. Further, the impact of strain variation on virulence, disease presentation, and mortality is not well understood. Therefore, this study evaluate and compare the virulence and disease progression of B. pseudomallei strains K96243 and HBPUB10303a, following aerosol challenge in a standardized BALB/c mouse model of infection. The natural history analysis of disease progression monitored conditions such as weight, body temperature, appearance, activity, bacteremia, organ and tissue colonization (pathological and histological analysis) and immunological responses. This study provides a detailed, direct comparison of infection with different B. pseudomallei strains and set up the basis for a standardized model useful to test different medical countermeasures against Burkholderia species. Further, this protocol serves as a guideline to standardize other bacterial aerosol models of infection or to define biomarkers of infectious processes caused by other intracellular pathogens.
Leukoencephalopathies are a group of white matter disorders related to abnormal formation, maintenance, and turnover of myelin in the central nervous system. These disorders of the brain are ...categorized according to neuroradiological and pathophysiological criteria. Herein, we have identified a unique form of leukoencephalopathy in seven patients presenting at ages 2 to 4 months with progressive microcephaly, spastic quadriparesis, and global developmental delay. Clinical, metabolic, and imaging characterization of seven patients followed by homozygosity mapping and linkage analysis were performed. Next generation sequencing, bioinformatics, and segregation analyses followed, to determine a loss of function sequence variation in the phospholipase A
-activating protein encoding gene (PLAA). Expression and functional studies of the encoded protein were performed and included measurement of prostaglandin E
and cytosolic phospholipase A
activity in membrane fractions of fibroblasts derived from patients and healthy controls. Plaa-null mice were generated and prostaglandin E
levels were measured in different tissues. The novel phenotype of our patients segregated with a homozygous loss-of-function sequence variant, causing the substitution of leucine at position 752 to phenylalanine, in PLAA, which causes disruption of the protein's ability to induce prostaglandin E
and cytosolic phospholipase A
synthesis in patients' fibroblasts. Plaa-null mice were perinatal lethal with reduced brain levels of prostaglandin E
The non-functional phospholipase A
-activating protein and the associated neurological phenotype, reported herein for the first time, join other complex phospholipid defects that cause leukoencephalopathies in humans, emphasizing the importance of this axis in white matter development and maintenance.
Pneumonia is a serious problem worldwide. We recently demonstrated that innate defense mechanisms of the lung are highly inducible against pneumococcal pneumonia. To determine the breadth of ...protection conferred by stimulation of lung mucosal innate immunity, and to identify cells and signaling pathways activated by this treatment, mice were treated with an aerosolized bacterial lysate, then challenged with lethal doses of bacterial and fungal pathogens. Mice were highly protected against a broad array of Gram-positive, Gram-negative, and class A bioterror bacterial pathogens, and the fungal pathogen, Aspergillus fumigatus. Protection was associated with rapid pathogen killing within the lungs, and this effect was recapitulated in vitro using a respiratory epithelial cell line. Gene expression analysis of lung tissue showed marked activation of NF-kappaB, type I and II IFN, and antifungal Card9-Bcl10-Malt1 pathways. Cytokines were the most strongly induced genes, but the inflammatory cytokines TNF and IL-6 were not required for protection. Lung-expressed antimicrobial peptides were also highly up-regulated. Taken together, stimulated innate resistance appears to occur through the activation of multiple host defense signaling pathways in lung epithelial cells, inducing rapid pathogen killing, and conferring broad protection against virulent bacterial and fungal pathogens. Augmentation of innate antimicrobial defenses of the lungs might have therapeutic value for protection of patients with neutropenia or impaired adaptive immunity against opportunistic pneumonia, and for defense of immunocompetent subjects against a bioterror threat or epidemic respiratory infection.
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