Search

Aortico-left ventricular tunnel (ALVT) is a rare congenital disease characterised by extracardiac channel communication between the ascending aorta and the ventricle. However, there have been no reports of ALVT compressing the coronary arteries. Here, we report the case of a 57-year-old woman who presented with unstable angina due to right coronary artery compression caused by a giant aneurysmal ALVT. Preoperative imaging failed to accurately diagnose this rare anomaly. Finally, we combined surgical exploration and a three-dimensional reconstruction technique to diagnose ALVT. After surgical resection of the aneurysm and repair of the tunnel, the patient's angina resolved. This case illustrates the importance of recognising extrinsic compression of the coronary artery as a potential cause of angina pectoris. It also highlights that ALVT with atypical anatomical features may require multiple imaging techniques and even surgical exploration to confirm the diagnosis., 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., (© 2024 The Authors.)

Surface-enhanced Raman scattering (SERS) is renowned for amplifying Raman signals, with electromagnetic mechanism (EM) enhancement arising from localized surface plasmon resonances and chemical mechanism (CM) enhancement as a result of charge transfer interactions. Despite the conventional emphasis on EM as a result of plasmonic effects, recent findings highlight the significance of CM when noble metals appear as smaller entities. However, the threshold size of the noble metal clusters/particles corresponding to the switch in SERS mechanisms is not clear at present. In this work, the VSe 2- x O x /Au composites with different Au sizes are employed, in which a clear view of the SERS mechanism switch is observed at the Au size range of 16-21 nm. Our findings not only provide insight into the impact of noble metal size on SERS efficiency but also offer quantitative data to assist researchers in making informed judgments when analyzing SERS mechanisms.

Plasmonic materials can generate strong electromagnetic fields to boost the Raman scattering of surrounding molecules, known as surface-enhanced Raman scattering. However, these electromagnetic fields are heterogeneous, with only molecules located at the 'hotspots', which account for ≈ 1% of the surface area, experiencing efficient enhancement. Herein, we propose patterned plasmonic trimers, consisting of a pair of plasmonic dimers at the bilateral sides and a trap particle positioned in between, to address this challenge. The trimer configuration selectively directs probe molecules to the central traps where 'hotspots' are located through chemical affinity, ensuring a precise spatial overlap between the probes and the location of maximum field enhancement. We investigate the Raman enhancement of the Au@Al 2 O 3 -Au-Au@Al 2 O 3 trimers, achieving a detection limit of 10 -14  M of 4-methylbenzenethiol, 4-mercaptopyridine, and 4-aminothiophenol. Moreover, single-molecule SERS sensitivity is demonstrated by a bi-analyte method. Benefiting from this sensitivity, our approach is employed for the early detection of lung tumors using fresh tissues. Our findings suggest that this approach is sensitive to adenocarcinoma but not to squamous carcinoma or benign cases, offering insights into the differentiation between lung tumor subtypes., (© 2024. The Author(s).)

Plasmon-induced oxidation has conventionally been attributed to the transfer of plasmonic hot holes. However, this theoretical framework encounters challenges in elucidating the latest experimental findings, such as enhanced catalytic efficiency under uncoupled irradiation conditions and superior oxidizability of silver nanoparticles. Herein, we employ liquid surface-enhanced Raman spectroscopy (SERS) as a real-time and in situ tool to explore the oxidation mechanisms in plasmonic catalysis, taking the decarboxylation of p -mercaptobenzoic acid (PMBA) as a case study. Our findings suggest that the plasmon-induced oxidation is driven by reactive oxygen species (ROS) rather than hot holes, holding true for both the Au and Ag nanoparticles. Subsequent investigations suggest that plasmon-induced ROS may arise from hot carriers or energy transfer mechanisms, exhibiting selectivity under different experimental conditions. The observations were substantiated by investigating the cleavage of the carbon-boron bonds. Furthermore, the underlying mechanisms were clarified by energy level theories, advancing our understanding of plasmonic catalysis.

Deep venous thrombosis (DVT) is the third most common cardiovascular disease. Its clinical therapeutic effect is unsatisfactory due to the high rate of postthrombotic syndrome. Several studies have demonstrated the involvement of miRNAs in DVT. Therefore, we identified differentially expressed miRNAs in patients with DVT and explored their effects and underlying mechanism on endothelial cell (EC) injury.Differentially expressed miRNAs were identified via microRNA sequencing and verified using real-time quantitative PCR. The biological function of miR-181c-5p in human umbilical vein endothelial cell (HUVEC) injury stimulated by oxidized low-density lipoprotein (ox-LDL) was investigated. The target gene of miR-181c-5p was analyzed using bioinformatics and verified via dual-luciferase reporter assay.miRNA sequencing showed that miR-181c-5p was downregulated in the peripheral blood of patients with DVT. Furthermore, miR-181c-5p had a high clinical diagnostic value for DVT by receiver operating characteristic curve analysis. An in vitro cell model of EC injury, miR-181c-5p, was repressed in ox-LDL-treated HUVECs. Enhancing miR-181c-5p expression could alleviate the inhibition cell viability, cell apoptosis, raising ROS and MDA production, the reducing SOD level, and the elevated levels of thrombosis-related factor, ET-1 and vWF induced by ox-LDL. Further analysis revealed that FBJ osteosarcoma oncogene (FOS) is a target of miR-181c-5p and could antagonize the protective role of miR-181c-5p in ox-LDL-induced HUVEC injury.Our research demonstrated that miR-181c-5p could attenuate ox-LDL-induced EC injury and thrombosis-related factor expression by negatively regulating FOS. These findings suggest that the miR-181c-5p/FOS axis is a promising therapeutic target for DVT.

Tropical forests, where the soils are nitrogen (N) rich but phosphorus (P) poor, have a disproportionate influence on global carbon (C) and N cycling. While N deposition substantially alters soil C and N retention in tropical forests, whether P input can alleviate these N-induced effects by regulating soil microbial functions remains unclear. We investigated soil microbial taxonomy and functional traits in response to 10-year independent and interactive effects of N and P additions in a primary and a secondary tropical forest in Hainan Island. In the primary forest, N addition boosted oligotrophic bacteria and phosphatase and enriched genes responsible for C-, P-mineralization, nitrification and denitrification, suggesting aggravated P limitation while N excess. This might stimulate P excavation via organic matter mineralization, and enhance N losses, thereby increasing soil CO 2 and N 2 O emissions by 86% and 110%, respectively. Phosphorus and NP additions elevated C-mining enzymes activity mainly due to intensified C limitation, causing 82% increase in CO 2 emission. In secondary forest, P and NP additions reduced phosphatase activity, enriched fungal copiotrophs and increased microbial biomass, suggesting removal of nutrient deficiencies and stimulation of fungal growth. Meanwhile, soil CO 2 emission decreased by 25% and N 2 O emission declined by 52-82% due to alleviated P acquisition from organic matter decomposition and increased microbial C and N immobilization. Overall, N addition accelerates most microbial processes for C and N release in tropical forests. Long-term P addition increases C and N retention via reducing soil CO 2 and N 2 O emissions in the secondary but not primary forest because of strong C limitation to microbial N immobilization. Further, the seasonal and annual variations in CO 2 and N 2 O emissions should be considered in future studies to test the generalization of these findings and predict and model dynamics in greenhouse gas emissions and C and N cycling., 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 © 2023 Elsevier Ltd. All rights reserved.)

Elucidating the effects of atmospheric nitrogen (N) deposition on the photosynthetic capacity of plants is critical to understand forest growth and conservation under global change. However, studies on this topic generally consider only understory N addition, which ignores the effect of canopy interception. In this study, we conducted a field experiment in a subtropical forest to compare the effects of canopy vs. understory N addition on the photosynthetic rate of canopy and understory species. We found that canopy N addition enhanced the photosynthetic rate of canopy species by increasing leaf hydraulic conductivity and shortening the distance of CO 2 transportation. In contrast, understory N addition had non-significant effects on the photosynthetic rate of canopy species. Moreover, the photosynthetic rate of understory species was not affected by canopy or understory N addition. Interestingly, changes in hydraulic conductivity contributed more to accelerating the photosynthetic rate than changes in CO 2 transport distance. Our results provide important insights into the dissimilar effects of canopy and understory N addition on the photosynthetic rates of species in subtropical forests. Based on our findings, we highlighted the urgent need to consider canopy processes in future studies on N deposition., 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 © 2022 Wu, Chen, Zhou, Ye and Wu.)

The impacts of enhanced nitrogen (N) deposition on the global forest carbon (C) sink and other ecosystem services may depend on whether N is deposited in reduced (mainly as ammonium) or oxidized forms (mainly as nitrate) and the subsequent fate of each. However, the fates of the two key reactive N forms and their contributions to forest C sinks are unclear. Here, we analyze results from 13 ecosystem-scale paired 15 N-labelling experiments in temperate, subtropical, and tropical forests. Results show that total ecosystem N retention is similar for ammonium and nitrate, but plants take up more labelled nitrate ([Formula: see text]%) ([Formula: see text]) than ammonium ([Formula: see text]%) while soils retain more ammonium ([Formula: see text]%) than nitrate ([Formula: see text]%). We estimate that the N deposition-induced C sink in forests in the 2010s  is [Formula: see text] Pg C yr -1 , higher than previous estimates because of a larger role for oxidized N and greater rates of global N deposition., (© 2022. The Author(s).)

Understanding the successional process from a disturbed forest to a mature forest is essential for species recovery and conservation initiatives. The resource acquisition and drought tolerance of plants can be instructive to predictions of species abundance and distribution for different forests. However, they have not been adequately tested at different successional stages in karst regions. Here, we selected seven dominant species in an early-succession forest and 17 species in a late-succession forest in a karst region of southwestern China. Resource acquisition-related traits such as hydraulic conductivity and photosynthetic rate, and drought tolerance-related traits, including turgor loss point and wood density, were measured. We found that species in the early-succession forest had a higher hydraulic conductance and photosynthetic rate than those in the late-succession forest, while leaf water potential at turgor loss point and wood density showed nonsignificant differences between the two forests. In addition, we observed a significant negative relationship between photosynthetic rate and drought tolerance in the early-succession forest, which was not identified in late-succession forests. Our study indicates that resource acquisition rather than drought tolerance was the key factor explaining plant distributions in forests at different successional stages in karst regions. We also suggest that the resource acquisition and drought tolerance trade-off hypothesis is not always supported for karst region species. Our study could inform about the design of species replacements in successional forests and provide forest management and restoration guidelines for karst regions.

Organic waste, the predominant component of global solid waste, has never been higher, resulting in increased landfilling, incineration, and open dumping that releases greenhouse gases and toxins that contribute to global warming and environmental pollution. The need to create and adopt sustainable closed-loop systems for waste reduction and valorization is critical. Using organic waste as a feedstock, gasification and pyrolysis systems can produce biooil, syngas, and thermal energy, while reducing waste mass by as much as 85-95% through conversion into biochar, a valuable byproduct with myriad uses from soil conditioning to bioremediation and carbon sequestration. Here, we present a novel case study detailing the circular economy of gasification biochar in Singapore's Gardens by the Bay. Biochar produced from horticultural waste within the Gardens was tested as a partial peat moss substitute in growing lettuce, pak choi, and pansy, and found to be a viable substitute for peat moss. At low percentages of 20-30% gasification biochar, fresh weight yields for lettuce and pak choi were comparable to or exceeded those of plants grown in pure peat moss. The biochar was also analyzed as a potential additive to concrete, with a 2% biochar mortar compound found to be of suitable strength for non-structural functions, such as sidewalks, ditches, and other civil applications. These results demonstrate the global potential of circular economies based on local biochar creation and on-site use through the valorization of horticultural waste via gasification, generating clean, renewable heat or electricity, and producing a carbon-neutral to -negative byproduct in the form of biochar. They also indicate the potential of scaled-up pyrolysis or gasification systems for a circular economy in waste management., 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 © 2021 Elsevier B.V. All rights reserved.)

Parenterally administered rotavirus vaccines may overcome the low efficacy observed in resource-poor regions that use live oral formulations. We have reported work on a trivalent nonreplicating rotavirus vaccine (NRRV) for parenteral administration consisting of the recombinant tetanus toxoid P2 CD4 epitope fused to a truncated VP8* fragment (P2-VP8*) for the P[4], P[6], and P[8] serotypes of rotavirus adjuvanted with aluminum. An essential part of developing this vaccine candidate was devising quantification methods for each antigen in the trivalent NRRV in the presence of aluminum adjuvant. This report describes the development of quantitative inhibition enzyme-linked immunosorbent assays (ELISAs) for in vitro antigenicity determination of the adjuvanted trivalent NRRV using serotype-specific monoclonal antibodies (mAbs) against each of the P2-VP8* antigens. Adjuvanted trivalent vaccine samples are titrated and incubated with a constant concentration of specific mAbs against each NRRV P2-VP8* antigen variant. Unbound mAbs are measured by ELISA to indirectly quantify the amount of each antigen present in the trivalent vaccine. Sensitive, specific, and reproducible inhibition ELISAs were developed and qualified for each antigen and used for final product quantification and release testing without desorption of the vaccine antigen., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The effects of nitrogen (N) deposition on forests largely depend on its fate after entering the ecosystem. While several studies have addressed the forest fate of N deposition using 15 N tracers, the long-term fate and redistribution of deposited N in tropical forests remains unknown. Here, we applied 15 N tracers to examine the fates of deposited ammonium ( NH 4 + ) and nitrate ( NO 3 - ) separately over 3 years in a primary and a secondary tropical montane forest in southern China. Three months after 15 N tracer addition, over 60% of 15 N was retained in the forests studied. Total ecosystem retention did not change over the study period, but between 3 months and 3 years following deposition 15 N recovery in plants increased from 10% to 19% and 13% to 22% in the primary and secondary forests, respectively, while 15 N recovery in the organic soil declined from 16% to 2% and 9% to 2%. Mineral soil retained 50% and 35% of 15 N in the primary and secondary forests, with retention being stable over time. The total ecosystem retention of the two N forms did not differ significantly, but plants retained more 15 NO 3 - than 15 NH 4 + and the organic soil more 15 NH 4 + than NO 3 - . Mineral soil did not differ in 15 NH 4 + and 15 NO 3 - retention. Compared to temperate forests, proportionally more 15 N was distributed to mineral soil and plants in these tropical forests. Overall, our results suggest that atmospherically deposited NH 4 + and NO 3 - is rapidly lost in the short term (months) but thereafter securely retained within the ecosystem, with retained N becoming redistributed to plants and mineral soil from the organic soil. This long-term N retention may benefit tropical montane forest growth and enhance ecosystem carbon sequestration., (© 2021 John Wiley & Sons Ltd.)

Biochar, produced as a by-product of pyrolysis/gasification of waste biomass, shows great potential to reduce the environment impact, address the climate change issue, and establish a circular economy model. Despite the promising outlook, the research on the benefits of biochar remains highly debated. This has been attributed to the heterogeneity of biochar itself, with its inherent physical, chemical and biological properties highly influenced by production variables such as feedstock types and treating conditions. Hence, to enable meaningful comparison of results, establishment of an agreed international standard to govern the production of biochar for specific uses is necessary. In this study, we analyzed four key uses of biochar: 1) in agriculture and horticulture, 2) as construction material, 3) as activated carbon, and 4) in anaerobic digestion. Then the guidelines for the properties of biochar, especially for the concentrations of toxic heavy metals, for its environmental friendly application were proposed in the context of Singapore. The international status of the biochar industry code of practice, feedback from Singapore local industry and government agencies, as well as future perspectives for the biochar industry were explained., 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 © 2020 Elsevier B.V. All rights reserved.)

More effective rotavirus vaccines are essential for preventing extensive diarrheal morbidity and mortality in children under five years of age in low-resource regions. Nonreplicating rotavirus vaccines (NRRV) administered parenterally provide an alternate vaccination method to the current licensed oral vaccine. Live attenuated vaccines and may generate increased efficacy in low-resource settings because the parenteral administration route bypasses some of the challenges associated with oral administration, including differences in intestinal environments. Work described here supports development of a trivalent NRRV vaccine for parenteral administration to avoid complications of the gastrointestinal route. Recombinant VP8* subunit proteins representing some of the most prevalent strains of rotavirus infecting humans - DS-1 (P[4]), 1076 (P[6]), and Wa (P[8]) - were combined with an aluminum adjuvant and the P2 epitope of tetanus toxoid to enhance the immune response to this NRRV antigen. Vaccine formulation development included selection of aluminum hydroxide (Alhydrogel®) as an appropriate adjuvant as well as an optimal buffer to maintain antigen stability and optimize antigen binding to the adjuvant. Characterization assays were used to select the lead vaccine formulation and monitor formulation stability. The NRRV liquid formulation was stable for one year at 2°C to 8°C and four weeks at 37°C. Immunogenicity of the NRRV formulation was evaluated using a guinea pig model, where we demonstrated that the adjuvant provided a 20-fold increase in neutralization titer against a homologous antigen and that the P2-fusion also enhanced the serum neutralizing antibody responses. This vaccine candidate is currently being evaluated in human clinical trials.

Histone demethylases are promising therapeutic targets as they play fundamental roles for survival of Mixed lineage leukemia rearranged acute leukemia (MLLr AL). Here we focused on the catalytic Jumonji domain of histone H3 lysine 9 (H3K9) demethylase JMJD1C to screen for potential small molecular modulators from 149,519 natural products and 33,765 Chinese medicine components via virtual screening. JMJD1C Jumonji domain inhibitor 4 (JDI-4) and JDI-12 that share a common structural backbone were detected within the top 15 compounds. Surface plasmon resonance analysis showed that JDI-4 and JDI-12 bind to JMJD1C and its family homolog KDM3B with modest affinity. In vitro demethylation assays showed that JDI-4 can reverse the H3K9 demethylation conferred by KDM3B. In vivo demethylation assays indicated that JDI-4 and JDI-12 could induce the global increase of H3K9 methylation. Cell proliferation and colony formation assays documented that JDI-4 and JDI-12 kill MLLr AL and other malignant hematopoietic cells, but not leukemia cells resistant to JMJD1C depletion or cord blood cells. Furthermore, JDI-16, among multiple compounds structurally akin to JDI-4/JDI-12, exhibits superior killing activities against malignant hematopoietic cells compared to JDI-4/JDI-12. Mechanistically, JDI-16 not only induces apoptosis but also differentiation of MLLr AL cells. RNA sequencing and quantitative PCR showed that JDI-16 induced gene expression associated with cell metabolism; targeted metabolomics revealed that JDI-16 downregulates lactic acids, NADP + and other metabolites. Moreover, JDI-16 collaborates with all-trans retinoic acid to repress MLLr AML cells. In summary, we identified bona fide JMJD1C inhibitors that induce preferential death of MLLr AL cells., (© 2019 UICC.)

Denitrification is a major process contributing to the removal of nitrogen (N) from ecosystems, but its rate is difficult to quantify. The natural abundance of isotopes can be used to identify the occurrence of denitrification and has recently been used to quantify denitrification rates at the ecosystem level. However, the technique requires an understanding of the isotopic enrichment factor associated with denitrification, which few studies have investigated in forest soils. Here, soils collected from two tropical and two temperate forests in China were incubated under anaerobic or aerobic laboratory conditions for two weeks to determine the N and oxygen (O) isotope enrichment factors during denitrification. We found that at room temperature (20°C), NO 3 - was reduced at a rate of 0.17 to 0.35μgNg -1 h -1 , accompanied by the isotope fractionation of N ( 15 ε) and O ( 18 ε) of 31‰ to 65‰ (48.3±2.0‰ on average) and 11‰ to 39‰ (18.9±1.7‰ on average), respectively. The N isotope effects were, unexpectedly, much higher than reported in the literature for heterotrophic denitrification (typically ranging from 5‰ to 30‰) and in other environmental settings (e.g., groundwater, marine sediments and agricultural soils). In addition, the ratios of Δδ 18 O:Δδ 15 N ranged from 0.28 to 0.60 (0.38±0.02 on average), which were lower than the canonical ratios of 0.5 to 1 for denitrification reported in other terrestrial and freshwater systems. We suggest that the isotope effects of denitrification for soils may vary greatly among regions and soil types and that gaseous N losses may have been overestimated for terrestrial ecosystems in previous studies in which lower fractionation factors were applied., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bacteria are the highest abundant microorganisms in the soil. To investigate bacteria community structures, diversity, and functions, contrasting them in four different seasons all the year round with/within two different forest type soils of China. We analyzed soil bacterial community based on 16S rRNA gene sequencing via Illumina HiSeq platform at a temperate deciduous broad-leaved forest (Baotianman, BTM) and a tropical rainforest (Jianfengling, JFL). We obtained 51,137 operational taxonomic units (OTUs) and classified them into 44 phyla and 556 known genera, 18.2% of which had a relative abundance >1%. The composition in each phylum was similar between the two forest sites. Proteobacteria and Acidobacteria were the most abundant phyla in the soil samples between the two forest sites. The Shannon index did not significantly differ among the four seasons at BTM or JFL and was higher at BTM than JFL in each season. The bacteria community at both BTM and JFL showed two significant ( P < 0.05) predicted functions related to carbon cycle (anoxygenic photoautotrophy sulfur oxidizing and anoxygenic photoautotrophy) and three significant ( P < 0.05) predicted functions related to nitrogen cycle (nitrous denitrificaton, nitrite denitrification, and nitrous oxide denitrification). We provide the basis on how changes in bacterial community composition and diversity leading to differences in carbon and nitrogen cycles at the two forests.

According to manufacturers, inactivated poliovirus vaccines (IPVs) are freeze sensitive and require storage between 2°C and 8°C, whereas oral poliovirus vaccine requires storage at -20 °C. Introducing IPV into ongoing immunization services might result in accidental exposure to freezing temperatures and potential loss of vaccine potency. To better understand the effect of freezing IPVs, samples of single-dose vaccine vials from Statens Serum Institut (VeroPol) and multi-dose vaccine vials from Sanofi Pasteur (IPOL) were exposed to freezing temperatures mimicking what a vaccine vial might encounter in the field. D-antigen content was measured to determine the in vitro potency by ELISA. Immunogenicity testing was conducted for a subset of exposed IPVs using the rat model. Freezing VeroPol had no detectable effect on in vitro potency (D-antigen content) in all exposures tested. Freezing of the IPOL vaccine for 7 days at -20 °C showed statistically significant decreases in D-antigen content by ELISA in poliovirus type 1 (p < 0.0001) and type 3 (p = 0.048). Reduction of poliovirus type 2 potency also approached significance (p = 0.062). The observed loss in D-antigen content did not affect immunogenicity in the rat model. Further work is required to determine the significance of the loss observed and the implications for vaccine handling policies and practices., (Copyright © 2018. Published by Elsevier Ltd.)

Double mutant heat-labile toxin (dmLT) is a promising adjuvant for oral vaccine administration. The aims of our study were to develop sensitive methods to detect low concentrations of dmLT and to use the assays in preformulation studies to determine whether dmLT remains stable under conditions encountered by an oral vaccine. We developed a sandwich ELISA specific for intact dmLT and a sensitive SDS-PAGE densitometry method, and tested stability of dmLT in glass and plastic containers, in saliva, at the pH of stomach fluid, and in high-osmolarity buffers. The developed ELISA has a quantification range of 62.5 to 0.9ng/mL and lower limit of detection of 0.3ng/mL; the limit of quantification of the SDS-PAGE is 10μg/mL. This work demonstrates the application of dmLT assays in preformulation studies to development of an oral vaccine containing dmLT. Assays reported here will facilitate the understanding and use of dmLT as an adjuvant., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

An influenza pandemic remains a major public health concern. A key strategy to prevent a pandemic is to stockpile and pre-position stable influenza vaccine to allow rapid deployment in response to an outbreak. However, most influenza vaccines today are formulated as liquids that are stable only within a temperature range of 2°C to 8°C and require use of a cold chain, making vaccine transportation, distribution, and storage complicated and expensive, particularly for developing countries. To support the National Strategy for Pandemic Influenza preparedness in the United States and internationally, we developed two lead dry formulations of stable H1N1 influenza subunit vaccines using freeze-drying technology. The stable formulations contain an excipient combination of a disaccharide, such as sucrose or trehalose, and glycine, in addition to a surfactant and phosphate buffer. The freeze-dried vaccines were shown to be safe and remained immunogenic in an in vivo study in mice. Moreover, the lead formulations demonstrated no significant loss of activity after 40 months at storage temperatures of 25°C and 37°C. This stability can be particularly attractive as it could eliminate the need to use a cold chain for vaccine deployment and facilitate integration of vaccine distribution with general drug distribution where appropriate. These freeze-dried thermostable influenza subunit vaccines could also reduce the frequency of vaccine stockpile turnover, offering a cost-effective option for pandemic preparedness., Competing Interests: We have the following interests. Novartis Pharmaceuticals (East Hanover, NJ) provided the generous supply of two lots of influenza H1N1 subunit bulk vaccine for this study. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Vaccination is the most effective means of preventing influenza. However, the cost of producing annual seasonal influenza vaccines puts them out of reach for most developing countries. While live attenuated influenza vaccines are among the most efficacious and can be manufactured at low cost, they may require lyophilization to be stable enough for developing-country use, which adds a significant cost burden. The development of a liquid live attenuated seasonal influenza vaccine that is stable for around a year-the duration of an annual influenza season-would significantly improve not only the production output but also the use and accessibility of influenza vaccines in low-resource settings. In this study, potential stabilizing excipients were screened and optimized using the least stable influenza vaccine strain presently known, H1N1 (A/California/07/2009), as a model. The stability-conferring properties of the lead formulations were also tested with a Type B strain of influenza virus (B/Brisbane/60/2008). Stability was also evaluated with higher titers of influenza virus and exposure to agitation and freeze-thaw stresses to further confirm the stability of the lead formulations. Through this process, we identified a liquid formulation consisting of sucrose phosphate glutamate buffer with 1% arginine and 0.5% recombinant human serum albumin that provided storage stability of one year at 2-8°C for the influenza A and B strains tested., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Rotavirus infection, which can be prevented by vaccination, is responsible for a high burden of acute gastroenteritis disease in children, especially in low-income countries. An appropriate formulation, packaging, and delivery device for oral rotavirus vaccine has the potential to reduce the manufacturing cost of the vaccine and the logistical impact associated with introduction of a new vaccine, simplify the vaccination procedure, and ensure that the vaccine is safely and accurately delivered to children. Single-dose prefilled presentations can be easy to use; however, they are typically more expensive, can be a bottleneck during production, and occupy a greater volume per dose vis-à-vis supply chain storage and medical waste disposal, which is a challenge in low-resource settings. Multi-dose presentations used thus far have other issues, including increased wastage of vaccine and the need for separate delivery devices. In this study, the goals were to evaluate both the technical feasibility of using preservatives to develop a liquid multi-dose formulation and the primary packaging alternatives for orally delivered, liquid rotavirus vaccines. The feasibility evaluation included evaluation of commonly used preservatives for compatibility with rotavirus vaccines and stability testing of rotavirus vaccine in various primary containers, including Lameplast's plastic tubes, BD's oral dispenser version of Uniject™ (Uniject DP), rommelag's blow-fill-seal containers, and MEDInstill's multi-dose vial and pouch. These presentations were compared to a standard glass vial. The results showed that none of the preservatives tested were compatible with a live attenuated rotavirus vaccine because they had a detrimental effect on the viability of the virus. In the presence of preservatives, vaccine virus titers declined to undetectable levels within 1 month. The vaccine formulation without preservatives maintained a stability profile over 12 months in all primary containers that was similar to its profile in standard glass vials. This study demonstrates that there are multiple options for the primary container for rotavirus vaccines intended for oral delivery. Selection of an optimal primary container should take into consideration additional factors, including stability as well as cold chain volume, usability, cost, and manufacturing feasibility., (Copyright © 2016. Published by Elsevier Ltd.)

Denitrification removes fixed nitrogen (N) from the biosphere, thereby restricting the availability of this key limiting nutrient for terrestrial plant productivity. This microbially driven process has been exceedingly difficult to measure, however, given the large background of nitrogen gas (N2) in the atmosphere and vexing scaling issues associated with heterogeneous soil systems. Here, we use natural abundance of N and oxygen isotopes in nitrate (NO3 (-)) to examine dentrification rates across six forest sites in southern China and central Japan, which span temperate to tropical climates, as well as various stand ages and N deposition regimes. Our multiple stable isotope approach across soil to watershed scales shows that traditional techniques underestimate terrestrial denitrification fluxes by up to 98%, with annual losses of 5.6-30.1 kg of N per hectare via this gaseous pathway. These N export fluxes are up to sixfold higher than NO3 (-) leaching, pointing to widespread dominance of denitrification in removing NO3 (-) from forest ecosystems across a range of conditions. Further, we report that the loss of NO3 (-) to denitrification decreased in comparison to leaching pathways in sites with the highest rates of anthropogenic N deposition.

Purpose: Stable vaccines with long shelf lives and reduced dependency on the cold chain are ideal for stockpiling and rapid deployment during public emergencies, including pandemics. Spray drying is a low-cost process that has potential to produce vaccines stable at a wide range of temperatures. Our aim was to develop a stable formulation of a recombinant H1N1 influenza hemagglutinin vaccine candidate and take it to pilot-scale spray-drying production., Methods: Eight formulations containing different excipients were produced and assayed for antigen stability, powder characteristics, and immunogenicity after storage at a range of temperatures, resulting in the identification of four promising candidates. A pilot-scale spray-drying process was then developed for further testing of one formulation., Results: The pilot-scale process was used to reproducibly manufacture three batches of the selected formulation with yields >90%. All batches had stable physical properties and in vitro potency for 6 months at temperatures from -20°C to +50°C. Formulations stored for 3 months elicited immunogenic responses in mice equivalent to a frozen lot of bulk vaccine used as a stability control., Conclusions: This study demonstrates the feasibility of stabilizing subunit vaccines using a spray-drying process and the suitability of the process for manufacturing a candidate product.

The recent outbreak of H7N9 influenza virus infections in humans in China has raised concerns about the pandemic potential of this strain. Here, we test the efficacy of H3 stalk-based chimeric hemagglutinin universal influenza virus vaccine constructs to protect against H7N9 challenge in mice. Chimeric hemagglutinin constructs protected from viral challenge in the context of different administration routes as well as with a generic oil-in-water adjuvant similar to formulations licensed for use in humans.

Inadvertent vaccine freezing often occurs in the cold chain and may cause damage to freeze‑sensitive vaccines. Liquid vaccines that contain aluminum salt adjuvants are particularly vulnerable. Polyol cryoprotective excipients have been shown to prevent freeze damage to hepatitis B vaccine. In this study, we examined the freeze-protective effect of propylene glycol on diphtheria-tetanus-pertussis-whole-cell (DTwP) and acellular (DTaP) vaccines. Pilot lots of DTwP and DTaP formulated with 7.5% propylene glycol underwent 3 freeze-thaw treatments. The addition of propylene glycol had no impact on pH, particle size distribution, or potency of the vaccines prior to freeze-thaw treatment; the only change noted was an increase in osmolality. The potencies and the physical properties of the vaccines containing cryoprotectant were maintained after freeze-thawing and for 3 months in accelerated stability studies. The results from this study indicate that formulating vaccines with propylene glycol can protect diphtheria-tetanus-pertussis vaccines against freeze damages.

Administering vaccines directly to mucosal surfaces can induce both serum and mucosal immune responses. Mucosal responses may prevent establishment of initial infection at the port of entry and subsequent dissemination to other sites. The sublingual route is attractive for mucosal vaccination, but both a safe, potent adjuvant and a novel formulation are needed to achieve an adequate immune response. We report the use of a thermoresponsive gel (TRG) combined with a double mutant of a bacterial heat-labile toxin (dmLT) for sublingual immunization with a trivalent inactivated poliovirus vaccine (IPV) in mice. This TRG delivery system, which changes from aqueous solution to viscous gel upon contact with the mucosa at body temperature, helps to retain the formulation at the site of delivery and has functional adjuvant activity from the inclusion of dmLT. IPV was administered to mice either sublingually in the TRG delivery system or intramuscularly in phosphate-buffered saline. We measured poliovirus type-specific serum neutralizing antibodies as well as polio-specific serum Ig and IgA antibodies in serum, saliva, and fecal samples using enzyme-linked immunosorbent assays. Mice receiving sublingual vaccination via the TRG delivery system produced both mucosal and serum antibodies, including IgA. Intramuscularly immunized animals produced only serum neutralizing and binding Ig but no detectable IgA. This study provides proof of concept for sublingual immunization using the TRG delivery system, comprising a thermoresponsive gel and dmLT adjuvant.

Vaccination is considered the most cost-effective approach to preventing infectious diseases, yet better formulations and delivery methods for efficient distribution and administration of vaccines are needed, especially for low-resource settings. A fast-dissolving tablet (FDT) that could be packaged in a compact stackable blister sheet is a potentially attractive option for formulating oral vaccines, since it would minimally impact the cold chain and could potentially be administered directly to patients without reconstitution. This study focused on using one component of a live attenuated trivalent vaccine under development to produce a FDT for the prevention of diarrhea induced by enterotoxigenic Escherichia coli (ETEC). Ten formulations were prepared and freeze dried to produce FDTs. Three freezing conditions were explored, along with different drying and package sealing methods. Physical properties examined included structural integrity, dissolution time, moisture content, and glass transition temperature. Bacterial viability was tested by assaying for colony-forming units. The formulation compositions and freeze-drying parameters were adjusted in an iterative process to arrive at a promising formulation for the ETEC vaccine tablet. This formulation included sucrose and trehalose as cryoprotectants; phosphate and glutamate salts as buffers and stabilizers; and Natrosol(®), polyvinylpyrrolidone, and mannitol as binders. The process loss in viability during freeze drying was less than 0.3 log 10 (50% recovery) for the optimized vaccine tablet formulation. The final tablets were robust, disintegrated in less than 10s, and preserved the bacteria at 2-8°C for at least 12 months with less than 0.4 log 10 loss (40% recovery) in viability during storage. This study indicates that the FDT produced by freeze drying directly in a blister sheet could be a practical option for formulating ETEC vaccines for oral immunization and help to facilitate delivery of lifesaving vaccines, particularly in low-resource settings., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

New vaccine platform and delivery technologies that can have significant positive impacts on the effectiveness, acceptability, and safety of immunizations in developing countries are increasingly available. Although donor support for vaccine technology development is strong, the uptake of proven technologies by the vaccine industry and demand for them by purchasers continues to lag. This article explains the challenges and opportunities associated with accelerating the availability of innovative and beneficial vaccine technologies to meet critical needs in resource-poor settings over the next decade. Progress will require increased dialog between the public and private sectors around vaccine product attributes; establishment of specifications for vaccines that mirror programmatic needs; stronger encouragement of vaccine developers to consider novel technologies early in the product development process; broader facilitation of research and access to technologies through the formation of centers of excellence; the basing of vaccine purchase decisions on immunization systems costs rather than price per dose alone; possible subsidization of early technology adoption costs for vaccine producers that take on the risks of new technologies of importance to the public sector; and the provision of data to purchasers, better enabling them to make informed decisions that take into account the value of specific product attributes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A number of product development partnerships are actively developing new vaccines to combat infectious diseases in developing countries. To be effective, the products under development should not only be safe, efficacious, and affordable, but they should also have additional desirable technical attributes, including enhanced stability, efficient packaging, and improved ease of delivery. New technologies are now available to achieve these attributes; however, many of the technologies have yet to be adopted by the vaccine industry. This commentary discusses the opportunities and challenges associated with advancing such attributes, especially vaccine thermostability and dose sparing strategies, and the critical issues that must be addressed to bridge the gap between technology development and product development., (Copyright © 2012 Wiley Periodicals, Inc.)

Research on spray drying as a processing method to improve vaccine stabilization and to enable novel routes of vaccine delivery has produced promising results; however, the method has yet to be adopted for the manufacture of vaccine products by the pharmaceutical industry. This article reviews the status of spray-drying technology and discusses barriers and opportunities for its future application to vaccines.

All vaccines are susceptible to damage by elevated temperatures and many are also damaged by freezing. The distribution, storage, and use of vaccines therefore present challenges that could be reduced by enhanced thermostability, with resulting improvements in vaccine effectiveness. Formulation and processing technologies exist that can improve the stability of vaccines at temperature extremes, however, customization is required for individual vaccines and results are variable. Considerations affecting decisions about stabilization approaches include development cost, manufacturing cost, and the ease of use of the final product. Public sector agencies can incentivize vaccine developers to prioritize stabilization efforts through advocacy and by implementing policies that increase demand for thermostable vaccines., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aluminum-containing adjuvants have been used to enhance the immune response against killed, inactivated, and subunit antigens for more than seven decades. Nevertheless, we are only beginning to gain important insight as to what may be some very fundamental parameters for optimizing their use. For example, there is evidence that the conventional approach of maximizing antigen binding (amount and/or strength) may not result in an optimal immune response. Adsorption of antigen onto the adjuvant has recently been suggested to decrease the thermal stability of some antigens; however, whether adsorption-induced alterations to the structure and/or stability of the antigen have consequences for the elicited immune response is unclear. Finally, the thermal stability of vaccines with aluminum-containing adjuvants is not robust. Optimizing the stability of these vaccines requires an understanding of the freeze sensitivity of the adjuvant, freeze and heat sensitivity of the antigen in the presence of the adjuvant, and perhaps most important, how (or whether) various approaches to formulation can be used to address these instabilities. This review attempts to summarize recent findings regarding issues that may dictate the success of vaccines with aluminum-containing adjuvants., (Copyright © 2010 Wiley-Liss, Inc.)

HSV-2-gD2 DNA vaccine was precisely delivered to immunologically sensitive regions of the skin epithelia using dry-coated microprojection arrays. These arrays delivered a vaccine payload to the epidermis and the upper dermis of mouse skin. Immunomicroscopy results showed that, in 43 ± 5% of microprojection delivery sites, the DNA vaccine was delivered to contact with professional antigen presenting cells in the epidermal layer. Associated with this efficient delivery of the vaccine into the vicinity of the professional antigen presenting cells, we achieved superior antibody responses and statistically equal protection rate against an HSV-2 virus challenge, when compared with the mice immunized with intramuscular injection using needle and syringe, but with less than 1/10th of the delivered antigen., (Copyright © 2010 Elsevier B.V. All rights reserved.)

There is an unmet medical need for a prophylactic vaccine against herpes simplex virus (HSV). DNA vaccines and cutaneous vaccination have been tried for many applications, but few reports combine this vaccine composition and administration route. We compared DNA administration using the Nanopatch™, a solid microprojection device coated with vaccine comprised of thousands of short (110 μm) densly-packed projections (70 μm spacing), to standard intramuscular DNA vaccination in a mouse model of vaginal HSV-2 infection. A dose-response relationship was established for immunogenicity and survival in both vaccination routes. Appropriate doses administered by Nanopatch™ were highly immunogenic and enabled mouse survival. Vaginal HSV-2 DNA copy number day 1 post challenge correlated with survival, indicating that vaccine-elicited acquired immune responses can act quickly and locally. Solid, short, densely-packed arrays of microprojections applied to the skin are thus a promising route of administration for DNA vaccines., (Copyright © 2010. Published by Elsevier Ltd.)

Biometric inventories for 25 years, from 1983 to 2005, indicated that the Jianfengling tropical mountain rain forest in Hainan, China, was either a source or a modest sink of carbon. Overall, this forest was a small carbon sink with an accumulation rate of (0.56+/-0.22) Mg C ha(-1)yr(-1), integrated from the long-term measurement data of two plots (P9201 and P8302). These findings were similar to those for African and American rain forests ((0.62+/-0.23) Mg C ha(-1)yr(-1)). The carbon density varied between (201.43+/-29.38) Mg C ha(-1) and (229.16+/-39.2) Mg C ha(-1), and averaged (214.17+/-32.42) Mg C ha(-1) for plot P9201. Plot P8302, however, varied between (223.95+/-45.92) Mg C ha(-1) and (254.85+/-48.86) Mg C ha(-1), and averaged (243.35+/-47.64) Mg C ha(-1). Quadratic relationships were found between the strength of carbon sequestration and heavy rainstorms and dry months. Precipitation and evapotranspiration are two major factors controlling carbon sequestration in the tropical mountain rain forest.

A combination of unique stabilizers and mild spray drying process conditions was employed to produce heat-stable measles vaccine powder. Live attenuated measles vaccine from Serum Institute of India was formulated with pharmaceutically approved stabilizers, including sugars, proteins, amino acids, polymers, surfactants, and plasticizers, as well as charged ions. In addition, the effects of buffer salt and pH on the storage stability of measles virus were examined. The potency of the dried vaccine stored at several temperatures was quantified by TCID(50) assay on Vero cells. As a comparison to other process methods, lead formulations were also subjected to freeze drying and foam drying. The optimized measles vaccine formulation tested at 37 degrees C was stable for approximately 8 weeks (i.e. time for 1 log TCID(50) loss). The measles titer decreased in a bi-phasic manner, with initial rapid loss within the first week but relative stability thereafter. Key stabilizers identified during the formulation screening processes were L-arginine, human serum albumin, and a combination of divalent cations. Spray drying was identified as the optimal processing method for the preparation of dried vaccine, as it generally resulted in negligible process loss and comparable, if not better storage stability, with respect to the other processes. Processing methods and formulation components were developed that produced a measles vaccine stable for up to 8 weeks at 37 degrees C, which surpassed the WHO requirement for heat stability of 1 week at that temperature., ((c) 2009 Elsevier Ltd. All rights reserved.)

The purpose of the present study was to develop a formulation of recombinant hepatitis B vaccine with improved stability at elevated temperatures. A validated in vitro antigen reactivity assay was used to measure the stability of the vaccine. The formulation development focused on modification of the interactions between the antigen and aluminum hydroxide adjuvant and subsequent optimization of the ionic aqueous environment of the adsorbed vaccine. A formulation of hepatitis B vaccine containing 40 mM histidine and 40 mM phosphate at pH 5.2 had considerably improved stability at elevated temperatures as measured by the in vitro antigen reactivity assay. The formulation exhibited 9-week stability at 55 degrees C and was subsequently shown to be stable both at 37 degrees C and at 45 degrees C for at least 6 months based on the in vitro antigen reactivity and immunogenicity in mice. The formulation comprises only excipients which have a history of safe use in approved drug products. The new vaccine formulation has the potential to be used outside the cold chain for part of its shelf life. This may improve the immunization coverage, simplify the logistics for outreach immunization, and ensure the potency of the vaccine in areas where the cold chain is insufficient.

Cold chain requirements for vaccine storage and distribution are both economic and logistical burdens for immunization programs, especially those in lower-resource settings. Inadvertent exposure of vaccines to both heat and freezing temperatures within such cold chains are frequently occurring problems in both developing and industrialized countries. Here we report on a new hepatitis B vaccine formulation that is stable against repeated freezing at -20 degrees C and is also stable for 12 months at 37 degrees C. The thermostable vaccine contains all the components of the original vaccine plus 7.5% (v/v) propylene glycol, 40mM phosphate, and 40mM histidine with a final pH of 5.2. The propylene glycol is responsible for the freeze stability while the other components are essential for the heat stability. This formulation was found to be well tolerated in rabbits without any significant local or systemic side effects. The improved stability of this hepatitis B vaccine could be a key factor in ensuring vaccine effectiveness, extending immunization coverage, simplifying immunization logistics, and reducing the costs associated with the cold chain.

All vaccines lose potency over time and the rate of potency loss is temperature-dependent. Therefore, cold-chain systems have been established to ensure that the potency of vaccines is maintained by storing them under refrigerated conditions (in most cases between 2 and 8 degrees C) until the point of use. This article aims to review the approaches being used to develop thermostable vaccine formulations that would be resistant to damage caused by freezing or excessive heat, and that could reduce dependence on the cold chain. The challenges associated with the implementation of these novel formulations are discussed, as well as the potential benefits and opportunities of taking vaccines out of the cold chain.

Vaccines containing aluminum salt adjuvants are prone to inactivation following exposure to freeze-thaw stress. Many are also prone to inactivation by heat. Thus, for maximum potency, these vaccines must be maintained at temperatures between 2 degrees C and 8 degrees C which requires the use of the cold chain. Nevertheless, the cold chain is not infallible. Vaccines are subject to freezing during both transport and storage, and frozen vaccines are discarded (under the best circumstances) or inadvertently administered despite potentially reduced potency. Here we describe an approach to minimize our reliance on the proper implementation of the cold chain to protect vaccines from freeze-thaw inactivation. By including PEG 300, propylene glycol, or glycerol in a hepatitis B vaccine, particle agglomeration, changes in the fluorescence emission spectrum--indicative of antigen tertiary structural changes--and losses of in vitro and in vivo indicators of potency were prevented following multiple exposures to -20 degrees C. The effect of propylene glycol was examined in more detail and revealed that even at concentrations too low to prevent freezing at -10 degrees C, -20 degrees C, and -80 degrees C, damage to the vaccine could be prevented. A pilot study using two commercially available diphtheria, tetanus toxoid, and acellular pertussis (DTaP) vaccines suggested that the same stabilizers might protect these vaccines from freeze-thaw agglomeration as well. It remains to be determined if preventing agglomeration of DTaP vaccines preserves their antigenic activity following freeze-thaw events.

Recent studies have revealed that vaccines containing aluminum adjuvant are exposed to sub-zero temperatures while in the cold chain more frequently than was previously believed. This raises concerns that these freeze-sensitive vaccines may be damaged and offer inadequate protection. This study was undertaken to characterize the immediate qualitative changes of one such vaccine, hepatitis B, caused by freeze exposure. Hepatitis B vaccine was subjected to freezing temperatures ranging from 0 degrees C to -20 degrees C for up to three episodes with durations ranging from 1 hour to 7 days. The vaccine was analyzed for freezing point, particle size distribution, tertiary structure, and in vitro and in vivo potency. Whether or not hepatitis B vaccine freezes was shown to be dependent on an array of factors including temperature, rate of temperature change, duration of exposure, supercooling effects and vibration. Vaccine exposed to "mild" freezing (-4 degrees C or warmer) temperatures did not freeze and remained qualitatively unaltered. Single or repeated freezing events at temperatures of -10 degrees C or lower were associated with aggregation of the adjuvant-antigen particles, structural damage of the antigen, and reduction of immunogenicity in mice. Damage to the vaccine increased with duration of freezing, lower temperature, and the number of freezing episodes. With vibration, vaccine froze at -6 degrees C after 1 hour and damage occurred. Freezing and freeze damage to vaccines containing aluminum salt adjuvant represent real risks to the effectiveness of immunization and should be prevented by strengthening the cold chain system or, alternatively, development of freeze-stable vaccine formulations.

The purpose of this study was to develop a hepatitis-B surface antigen (HBsAg) dry powder vaccine formulation suitable for epidermal powder immunization (EPI) via an efficient, scalable powder-formation process. Several HBsAg dry powder formulations were prepared using four different powder-formation methods: freeze-drying/compress/grind/sieve (FD/C/G/S), spray-drying (SD), agarose beads, and spray freeze-drying (SFD). Powder properties and physical stability were determined using particle size analysis, tap density measurement, scanning electron microscopy, optical microscopy, and moisture content analysis. Physical, chemical and biochemical stability of HBsAg was determined by dynamic light scattering, an enzyme immune assay, and immunogenicity in a mouse or hairless guinea pig model. Out of the four powder-formation methods evaluated SFD outperformed other methods in the following considerations: good process efficiency, flexible scalability, and desirable particle characteristics for skin penetration. The stress posed by SFD appeared to be mild as HBsAg in the dry form retained its potency and immunogenicity. Notably, the mechanism of fast freezing by SFD actually promoted the preservation of HBsAg nanoparticle size, in good correlation with long-term biochemical stability. Among several formulations screened, the formulation containing 10 microg HBsAg in 1-mg powder with a tertiary mixture of trehalose, mannitol, and dextran, exhibited excellent overall stability performance. In conclusion, HBsAg dry powder formulations suitable for EPI were successfully prepared using SFD. Further, a systematic formulation development strategy allowed the development and optimization of an HBsAg dry powder formulation, demonstrating excellent long-term physical, biochemical, and immunological stability.

The purpose of this study was to evaluate whether a single peptide containing a major T cell epitope might induce peripheral tolerance in a complex allergen model. C57BL/6 mice were sensitized by intraperitoneal injection of house dust mite extract (HDM), and exposed to antigen via trachea instillation. Der p 1 peptide was administered by i.v. before or after sensitization. Lung lavage fluids were analyzed for cellular infiltration. Respiratory exposure of sensitized mice to antigen results in airway inflammation and eosinophilia. Intravenous administration of a single peptide protected sensitized mice from these changes. Further, the emergence of antigen-specific CD25(+)CD4+ and IL-10 secreting cell populations in DO11.10 mice was demonstrated after peptide administration. Thus, intravenous delivery of a single peptide epitope is capable of inducing peripheral tolerance and protection in a complex allergy model, possibly through regulatory T cells and bystander suppression.

Epidermal powder immunization (EPI) can efficiently deliver powdered protein vaccines to the epidermis. A phase I clinical trial was conducted to evaluate powdered trivalent influenza vaccine delivered using the PowderJect ND5.2 delivery system. Subjects received either Fluvirin IM injection (15 microg of each influenza strain), a single EPI vaccination (15 microg of each influenza strain) or two adjacent EPI (total of 30 microg of each influenza strain). Systemic reactogenicity was similar between control and EPI vaccines. Site reactions following EPI were primarily mild and self-limiting. Seroconversions, titer increases and geometric mean titers to all strains were equivalent or higher in EPI-immunized groups than in controls. Powdered influenza vaccine delivered by EPI is safe and elicits humoral immune responses in humans.

Background: Atopic disorders are associated with an imbalanced T(H) cell response biased toward a strong T(H)2 type, resulting in excessive production of IgE antibodies, eosinophil recruitment and activation, and mast cell degranulation. Restoring the T(H)1-T(H)2 balance by increasing the antigen-specific T(H)1 response has been pursued for specific allergy immunotherapy. Synthetic oligodeoxynucleotides containing unmethylated CG dinucleotides (CpG) are strong T(H)1 adjuvants and are being investigated for allergy immunotherapy., Objective: This study was designed to investigate the protective role of antigen-specific T(H)1 responses induced by epidermal powder immunization with ovalbumin (OVA) and CpG in a murine airway inflammation model., Methods: An allergy model was used in which BALB/c mice were sensitized and then challenged with OVA. Mice received prophylactic or therapeutic immunizations with OVA, CpG, or both. After challenge, pulmonary inflammation and cell infiltration were measured on the basis of BAL cell counts and lung histology. Immune response was determined by measuring the levels of lavage cytokines and serum antibodies., Results: Coadministration of OVA and CpG by means of subcutaneous injection or epidermal powder immunization, although inducing a strong T(H)1 response, neither suppressed T(H)2 cytokines nor offered protection against pulmonary eosinophilia and histopathology in a mouse challenge model. However, when CpG was used as a stand-alone treatment of previously sensitized animals, protection against allergic airway inflammation was observed. After challenge with OVA, eosinophilia was suppressed in the lungs of the CpG-treated mice., Conclusion: This finding argues against the approach of boosting an allergen-dependent T(H)1 response and favors induction of an antigen-independent T(H)1 response for allergy immunotherapy.

The purpose of this study was to develop a spray-freeze-drying (SFD) process for preparing an influenza vaccine dry powder formulation suitable for epidermal powder immunization. After preformulation of two types of flu vaccines, their dry-powder formulations were prepared by SFD. Powder properties and physical stability were determined using particle size analysis, tap density measurement, scanning electron microscopy, optical microscopy, and moisture content analysis. Chemical and biochemical stability of vaccine antigens was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, single radial immunodiffusion assay, and in vivo immunogenicity in a mouse model. We demonstrated that SFD could produce high-density particles-a critical parameter for effective skin penetration. From the stability perspective, the stress posed by SFD was mild because the antigen in the dry powder retained its stability, potency, and immunogenicity. Among several formulations screened, we noted that formulation composition has a significant role in the powder's long-term physical and biochemical stability. One formulation, in particular, containing sub-unit vaccine (45 microg of antigen in 1 mg of powder) with a tertiary mixture of trehalose, mannitol, and dextran, exhibited excellent overall stability, including acceptable biochemical stability after being exposed to a highly humid environment. After all, we have not only demonstrated the suitability of SFD to prepare powders for epidermal powder immunization but also developed a systematic formulation development strategy that allowed the optimization of an influenza vaccine dry powder formulation. More important, this study led to the selection of a formulation system that had been successfully tested in a human clinical study., (Copyright 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1912-1923, 2004)

Epidermal powder immunization (EPI) of mice with an influenza vaccine elicited consistently a higher hemagglutination inhibition (HI) antibody titers than intramuscular (IM) injection using the same dose of vaccine. The epidermal Langerhans cells (LCs) at the site of EPI were found to play an important role in the immune responses. Indeed, depletion of LCs from the immunization site prior to EPI caused a significant reduction in the antibody response. Transfer of LCs isolated from the EPI sites to naive mice induced a robust antigen-specific antibody response. Cytokines produced by target site cells appear to be important for the augmented immune responses induced by EPI. LTR72, a genetically detoxified heat-labile toxin from Escherichia coli with a strong adjuvant effect in EPI, was found to bind the keratinocytes of the epidermis, but not the LCs, and caused the production of elevated TNF-alpha and IL-12 cytokines in emigrating epidermal cells. These results have important implications for the development of a more efficacious human influenza vaccine.

Purpose: Fluid-bed spray-coating process is widely used to prepare non-protein pharmaceutical solid dosage forms using macro-size seed particles (200-1000 microm) at kilogram batch sizes. In this study we developed a small-scale fluid-bed spray-coating process (20 g) to produce micro-sized vaccine powder formulations (40-60 microm) for epidermal powder immunization (EPI) METHODS: A bench-top spray coater was used to spray two vaccines, diphtheria toxoid (dT) and alum-adsorbed hepatitis-B surface antigen (Alum-HBsAg), onto crystalline lactose particles of 40-60 microm in diameter. Particle properties such as particle size, surface morphology, and degree of particle agglomeration were determined. Protein stability was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immunogenicity of the vaccine was evaluated in vivo by needle injection and epidermal powder immunization (EPI) of mice or guinea pigs., Results: Coating feasibility was demonstrated for both vaccine formulations containing different excipients. However, the nature of the vaccine antigen appeared to affect coating feasibility in terms of particle agglomeration considerably. Delivery of spray-coated dT and alum-HBsAg through EPI to mice and guinea pigs, respectively, generated significant antibody responses, at a level comparable to liquid formulation delivered subcutaneously through needle/syringe injection., Conclusions: The new spray-coating process represents an important technical advance and may provide a useful tool for developing high-valued biopharmaceutical powder formulations for novel applications. The strong in vivo performance of the coated dT and alum-HBsAg powders by EPI further demonstrated that spray-coating is a viable dry powder formulation process and the skin's epidermal layer presents an efficient vaccine delivery route.