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Background: Dupilumab has been used with significant benefit in the treatment of asthma and chronic rhinosinusitis with nasal polyps (CRSwNP). Phase 3 clinical trials have demonstrated transient eosinophilia and rare eosinophil-related and other adverse effects., Objective: To characterize dupilumab-associated eosinophilia (absolute eosinophil count [AEC] ≥ 1.5 x 10 3 /μL within 36 weeks of dupilumab initiation) and adverse effects associated in real-world patients with asthma and CRSwNP in the United States., Methods: Retrospective chart review of 251 patients on dupilumab for asthma and/or CRSwNP seen at a single institution., Results: Among the 142 patients who had AECs checked before and after treatment, 16 (11.3%) patients had post-treatment eosinophilia, including 11 (7.7%) patients who had new eosinophilia upon dupilumab initiation. Thirteen patients with post-treatment eosinophilia remained on dupilumab, 10 of whom had resolution of eosinophilia. Eosinophil-related adverse effects were rare and cases of eosinophilic granulomatous polyangiitis (EGPA) were limited to 1 patient with eosinophilia and 1 patient with normal eosinophil levels on systemic steroids. Other adverse effects included arthralgias (13/251, 5.2%), rash (8/251, 3.2%), and conjunctivitis (7/251, 2.8%). All patients with pre-treatment eosinophilia and the majority of patients with post-treatment eosinophilia received significant treatment benefit for their respiratory disease with dupilumab., Conclusion: While dupilumab-associated eosinophilia is seen in a subset of patients, persistent eosinophilia or eosinophil-related adverse effects are rare. Furthermore, treatment benefit on dupilumab despite eosinophilia supports its continued use in both asthma and CRSwNP., (Copyright © 2024. Published by Elsevier Inc.)

Background: Chronic cough, defined as a cough lasting 8 or more weeks, affects up to 10% of adults. Refractory chronic cough (RCC) is a cough that is uncontrolled despite comprehensive investigation and treatment of comorbid conditions while unexplained chronic cough (UCC) is a cough with no identifiable cause despite extensive evaluation of comorbid conditions. RCC and UCC are often poorly controlled. Understanding individuals' lived experience of the symptoms and impacts of these conditions may guide therapeutic strategies., Objectives: The primary objectives of this study were to assess respondents' perceptions of the key symptoms of RCC and UCC and the impacts of RCC and UCC and their symptoms on well-being, health-related quality of life, work productivity, and social relationships., Design: Qualitative study., Methods: This study enrolled 30 adults with physician-diagnosed RCC or UCC. Two trained qualitative researchers conducted individual, in-depth telephone interviews using a semi-structured interview guide. Interviews were audio-recorded, transcribed, coded, and systematically analyzed to identify content themes., Results: A total of 15 respondents with RCC and 15 with UCC were included in the study. Many respondents had RCC or UCC for a long duration (median 9 years, range: 0-24). Half of the respondents reported having a coughing episode at least once daily. Only 40% of respondents reported that medication had improved their symptoms. In over half of the respondents, RCC or UCC hindered communication, caused embarrassment, frustration, and worry, and lowered quality of life. Perceptions of meaningful treatment benefits in RCC or UCC varied widely across respondents., Conclusion: RCC and UCC remained poorly managed in many individuals and were associated with a wide range of symptoms and cough triggers that hindered daily activities and reduced emotional well-being. Understanding individuals' lived experiences may inform the development of RCC and UCC therapeutic strategies.

Background: The diagnosis and management of chronic cough in primary care is challenging despite it being one of the most common chronic conditions. Objective: Clinical characterization of patients with new-onset chronic cough in the primary care setting. Methods: This was a retrospective study of adult patients (ages ≥ 18 years) with at least three visits with primary care providers (PCP) for new-onset cough, with at least 8 weeks between the first and third visits, within a tertiary-care center and affiliated clinics between January 1, 2010, and January 1, 2019 ( N = 174). We calculated the frequency of primary care visits, diagnostic testing, specialist referrals, and prescribed medications up to 18 months after the third visit with a PCP for cough. Results: Of 174 patients who met the criteria of new-onset chronic cough, >50% had four or more primary care visits related to cough. Despite that, 91 (52.3%) did not receive a referral to a specialist, and 41 (23.5%) did not receive an order for a chest radiograph during the evaluation of the chronic cough. Antibiotics and systemic corticosteroids were prescribed to 106 (61%) and 63 (36%) of the patients, respectively, and 20% were prescribed opiates. No patients were prescribed central-neuromodulating agents, and angiotensin-converting enzyme inhibitors were discontinued in 48% of the patients who were taking them (12/25). Conclusion: We found considerable heterogeneity and discrepancies with clinical guideline recommendations in patients who presented with new chronic cough. There is a substantial unmet need to study chronic cough in the primary care setting to inform important stakeholders.

The aim of this work was to study the reduction of pollution load in the textile industry effluent by lipase producing Nocardiopsis alba. Nocardiopsis alba was isolated from the soap-stock of an oil refinery located in North Gujarat, India. Lipase 2 (EC 3.1.1.3) protein sequence was identified from Nocardiopsis alba and the 3D model was predicted by SWISS Modeling. Lipolytic Nocardiopsis alba showed 84 ± 0.3 %, 75 ± 0.4 %, and 81 ± 0.3 % reduction of Chemical Oxygen Demand, Biochemical Oxygen Demand and Total Solids, respectively when was grown in textile industrial wastewater at 37 ± 1 °C, 120 rpm for 15 days. The findings of this research offer potential environmental benefits along with socio-economic benefits as well if implemented suitably., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Acute allergic reactions to messenger RNA (mRNA) vaccines are rare but may limit public health immunization efforts. Objectives: To characterize suspected allergic reactions to the first dose of coronavirus disease 2019 (COVID-19) mRNA vaccine and to assess the safety and utility of a two-step graded-dose protocol for the second dose of the Pfizer-BioNTech vaccine in patients with a history of low suspicion of anaphylaxis to their first dose. Methods: This was a retrospective evaluation of referrals to the allergy and immunology clinic for a presumed allergic reaction to the first dose of the COVID-19 mRNA vaccine (Pfizer-BioNTech or Moderna) between December 17, 2020, and February 28, 2021. Recommendations for the second dose and outcomes were evaluated by trained board-certified allergists. Results: Seventy-seven patients presented with a Pfizer-BioNTech reaction (56 [72.7%]) or with a Moderna reaction (21 [27.3%]). Most patients (69.7%) had symptom onset within 4 hours. Most commonly reported symptoms were cutaneous (51.9%), cardiovascular (48.1%), and respiratory (33.8%) symptoms. Recommendations included to proceed with the single dose (70.1%), two-step graded dose (19.5%), or deferral (10.4%). Twelve of 15 patients completed the second dose with a graded-dose protocol. Of these patients, five reported at least one or more similar symptoms as experienced with their first dose. Conclusion: Of the patients with presumed allergic reactions to their first dose of COVID-19 mRNA vaccine, most were able to safely receive the second dose. For those with a low suspicion of anaphylaxis, the two-step graded protocol with the Pfizer-BioNTech vaccine was well tolerated. A graded-dose protocol could be an effective strategy for second-dose vaccination in those who may otherwise defer the second dose.

Background: Acute exacerbations of chronic rhinosinusitis (AECRS) are associated with significant morbidity and decreased quality of life. There are sparse data assessing the real-world impact of biologics on AECRS. Objectives: We sought to determine the impact of type 2-targeting biologics on the frequency of medication use for AECRS episodes. Methods: Antibiotic and/or systemic corticosteroid courses for AECRS were identified in a retrospective study from November 2015 to February 2020, at a single academic health system. The estimated yearly rates for antibiotic and corticosteroid courses were evaluated before and after initiation of type 2 biologics. Results: One-hundred and sixty-five patients with chronic rhinosinusitis (CRS) had received either omalizumab (n = 12), mepolizumab (n = 42), benralizumab (n = 44), dupilumab (n = 61), or reslizumab (n = 6). Seventy percent had CRS with nasal polyps, and 30% had CRS without nasal polyps. All the patients had asthma. When all the biologics were combined, the estimated yearly rate for antibiotics for AECRS decreased from 1.34 (95% confidence interval [CI], 1.12-1.59) to 0.68 (95% CI, 0.52-0.88) with biologic use (49% reduction, p < 0.001). Those with frequent AECRS (three or more courses of antibiotics in the 1 year before biologic use) had a larger degree of reduction, with an estimated yearly rate of 4.15 (95% CI, 3.79-4.55) to 1.58 (95% CI, 1.06-2.35) with biologic use (n = 27; 62% reduction; p < 0.001). Within the total cohort, the estimated yearly rate for systemic corticosteroids for AECRS decreased from 1.69 (95% CI, 1.42-2.02) to 0.68 (95% CI, 0.53-0.88) with biologic use (60% reduction; p < 0.001). Conclusion: Type 2-targeting biologics reduced medication use for AECRS. This suggested that biologics may be a therapeutic option for patients with frequent AECRS.

Background: Immunoglobulin E-mediated food allergy (FA) affects children and adults with variable age of onset. Phenotype and quality of life (QoL) differences between childhood-onset FA (COFA) and adult-onset FA (AOFA) are not known., Objective: To identify phenotypic and QoL differences between AOFA and COFA., Methods: A cross-sectional study of adults (≥18 years old) seen at Northwestern Memorial HealthCare clinics between 2002 and 2017 with an International Classification of Diseases ninth and tenth revision diagnosis of FA. Subjects completed a FA history survey and a FA QoL questionnaire. FA characteristics and QoL scores were compared between groups., Results: Among 294 consented subjects, 202 had a clinical history consistent with labeled immunoglobulin E-mediated FA. The onset of FA symptoms occurred before age 18 years (COFA) in 80 subjects and after age 18 years in 122 (AOFA) subjects. Shellfish reactions were most common in AOFA-labeled subjects (28%), whereas tree nut reactions were the most common in COFA-labeled subjects (55%) compared with other triggers. Hives (68% vs 52%, P = .03), facial swelling (69% vs 50%, P = .009), wheezing (56% vs 29%, P < .001), and vomiting (41% vs 22%, P = .005) were more often observed in COFA compared with AOFA. Total QoL was significantly reduced in COFA compared with AOFA (3.6 vs 3.0, P = .003) along with specific domains related to the following: allergen avoidance and dietary restriction (3.7 vs 3.1, P = .006), emotional impact (3.9 vs 3.2, P = .003), and risk of accidental exposure (3.6 vs 2.8, P = .001)., Conclusion: There are differences in specific food triggers and symptoms in adult-onset and childhood-onset labeled FA. Adults labeled with childhood-onset FA have reduced QoL., (Copyright © 2021 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: The Centers for Disease Control and Prevention advises that patients with moderate to severe asthma belong to a high-risk group that is susceptible to severe coronavirus disease 2019 (COVID-19). However, the association between asthma and COVID-19 has not been well-established., Objective: The primary objective was to determine the prevalence of asthma among patients with COVID-19 in a major US health system. We assessed the clinical characteristics and comorbidities in asthmatic and nonasthmatic patients with COVID-19. We also determined the risk of hospitalization associated with asthma and/or inhaled corticosteroid use., Methods: Medical records of patients with COVID-19 were searched by a computer algorithm (March 1 to April 15, 2020), and chart review was used to validate the diagnosis of asthma and medications prescribed for asthma. All patients had PCR-confirmed COVID-19. Demographic and clinical features were characterized. Regression models were used to assess the associations between asthma and corticosteroid use and the risk of COVID-19-related hospitalization., Results: Of 1526 patients identified with COVID-19, 220 (14%) were classified as having asthma. Asthma was not associated with an increased risk of hospitalization (relative risk, 0.96; 95% CI, 0.77-1.19) after adjusting for age, sex, and comorbidities. The ongoing use of inhaled corticosteroids did not increase the risk of hospitalization in a similar adjusted model (relative risk, 1.39; 95% CI, 0.90-2.15)., Conclusions: Despite a substantial prevalence of asthma in our COVID-19 cohort, asthma was not associated with an increased risk of hospitalization. Similarly, the use of inhaled corticosteroids with or without systemic corticosteroids was not associated with COVID-19-related hospitalization., (Copyright © 2020. Published by Elsevier Inc.)

Advances in understanding the pathogenic mechanisms of both rhinitis and chronic rhinosinusitis have resulted in new treatment options, especially for chronic rhinosinusitis. A review of relevant medical and surgical clinical studies shows that intranasal corticosteroids, antihistamines, and allergen immunotherapy continue to be the best treatments for chronic rhinitis. Dupilumab is the first biologic approved for chronic rhinosinusitis with polyps. Omalizumab, mepolizumab, and benralizumab may have a future role in the treatment of chronic rhinosinusitis. Novel corticosteroid delivery devices such as an exhalation delivery system for fluticasone and bioabsorbable sinus implants provide enhanced and localized distribution of corticosteroids. Surgical management tailored to the underlying disease process improves clinical outcomes in chronic rhinosinusitis with or without nasal polyposis. Advances in the understanding of the heterogeneous nature of rhinitis and rhinosinusitis have resulted in more precise treatments. Improving the understanding of different endotypes should provide better knowledge to determine appropriate current and new therapies to treat these diseases., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Human coronaviruses (HCoV) are common causes of respiratory illnesses (RI) despite preexisting humoral immunity. Sera were obtained near the onset of RI and 3 to 4 weeks later as part of a prospective study of 200 subjects evaluated for RI from 2009 to 2013. Antibodies against common HCoV strains were measured by enzyme-linked immunosorbent assay and neutralization assay comparing older adults with cardiopulmonary diseases (99 subjects) to younger, healthy adults (101 subjects). Virus shedding was detected in respiratory secretions by polymerase chain reaction. Of 43 HCoV-associated illnesses, 15 (35%) occurred in 14 older adults (aged ≥60 years) and 28 (65%) in 28 younger adults (aged 21-40 years). Binding and neutralizing antibodies were higher in older adults. Only 16 (35.7%) of RI with increases in binding antibodies also had increases in neutralizing antibodies to HCoV. Increases in binding antibodies with RI were more frequent than increased neutralizing antibodies and virus shedding, and more frequent in younger compared to older adults. Functional neutralizing antibodies were not stimulated as often as binding antibodies, explaining in part a susceptibility to reinfection with HCoV. Monitoring binding antibodies may be more sensitive for the serologic detection of HCoV infections., (Published 2020. This article is a U.S. Governmaent work and is in the public domain in the USA.)

Background: Severe asthma can be a challenging disease to manage by the provider and by the patient, supported by evidence of increased health-care utilization by this population. Patients with severe asthma should be screened for comorbidities because these often contribute to poorly controlled asthma. The impact of comorbidities, however, are not completely understood., Objective: To review common comorbidities and their impact on severe asthma., Methods: A review of relevant clinical research studies that examined comorbidities in severe or difficult-to-treat asthma., Results: A number of comorbid diseases, including rhinitis, rhinosinusitis, gastroesophageal reflux, and obstructive sleep apnea, are associated with severe or difficult-to-treat asthma. If present and untreated, these conditions may adversely affect asthma control, quality of life, and/or lung function, despite adequate treatment with step-up asthma controller therapy., Conclusion: Treatable comorbidities are associated with severe and difficult-to-control asthma. Failure to recognize these comorbidities may divert appropriate care and increase disease burden. Assessment and management of these risk factors may contribute to improved asthma outcome; however, more investigation is needed to understand the relationship of comorbidities and asthma due to inconsistency in the findings., Competing Interests: Authors have no conflicts of interest to declare pertaining to this article

In the present study, europium (III) oxide (Eu 2 O 3 )-doped polystyrene (PS) polymer films were synthesized using a solution-casting technique for different filler levels. These films were irradiated with 5, 25 and 50 kGy γ doses and characterized using various techniques, viz. X-ray diffraction (XRD), and UV-visible and photoluminescence (PL) spectroscopies as a function of composition level and radiation dose. The UV-visible spectra indicated a decrease in the optical direct band gap of composite films with increasing concentrations of dopant and radiation dose. The band gaps of composites obtained using Tauc's equation were found to be 4.38, 4.37, 4.36 and 4.34 eV for 0, 1, 3 and 5% Eu 2 O 3 -doped PS respectively, while the band gaps of 5% Eu 2 O 3 -doped PS polymer films irradiated with 5, 25 and 50 kGy were found to be 4.30, 4.26 and 4.21 eV, respectively. Photoluminescence (PL) emission spectra showed the characteristic peaks of Eu 3+ at 595 nm, 612 nm and 617 nm with an excitation wavelength of 247 nm. The intensity of characteristic peaks of Eu 3+ was observed to increase with increasing filler concentration, while it was found to decrease with increasing radiation dose. The polymer under study may be useful in accidental dosimetry. As photoluminescence studies are carried out after a gap of 200 h from irradiation and PL emission of γ-irradiated polymer yielded 10 times emission when compared with non-irradiated polymer., (© 2018 John Wiley & Sons, Ltd.)

Human coronaviruses (HCoV) are RNA viruses that cause respiratory tract infections with viral replication of limited duration. The host and viral population heterogeneity could influence clinical phenotypes. Employing long RT-PCR with Illumina sequencing, we quantified the gene mutation load at 0.5% mutation frequency for the 4529 bp-domain spanning the Spike gene (4086 bp) of HCoV-OC43 in four upper respiratory clinical specimens obtained during acute illness. There were a total of 121 mutations for all four HCoV samples with the average number of mutations at 30.3 ± 10.2, which is significantly higher than that expected from the Illumina sequencing error rate. There were two mutation peaks, one at the 5' end and the other near position 1 550 in the S1 subunit. Two coronavirus samples were genotype B and two were genotype D, clustering with HCoV-OC43 strain AY391777 in neighbor-joining tree phylogenetic analysis. Nonsynonymous mutations were 76.1 ± 14% of mutation load. Although lower than other RNA viruses such as hepatitis C virus, HCoV-OC43 did exhibit quasi-species. The rate of nonsynonymous mutations was higher in the HCoV-OC43 isolates than in hepatitis C (HCV) virus genotype 1a isolates analyzed for comparison in this study. These characteristics of HCoV-OC43 may affect viral replication dynamics, receptor binding, antigenicity, evolution, transmission, and clinical illness., (© 2017 Wiley Periodicals, Inc.)

Generation of protective immunity at mucosal surfaces can greatly assist the host defense against pathogens which either cause disease at the mucosal epithelial barriers or enter the host through these surfaces. Although mucosal routes of immunization, such as intranasal and oral, are being intensely explored and appear promising for eliciting protective mucosal immunity in mammals, their application in clinical practice has been limited due to technical and safety related challenges. Most of the currently approved human vaccines are administered via systemic (such as intramuscular and subcutaneous) routes. Whereas these routes are acknowledged as being capable to elicit antigen-specific systemic humoral and cell-mediated immune responses, they are generally perceived as incapable of generating IgA responses or protective mucosal immunity. Nevertheless, currently licensed systemic vaccines do provide effective protection against mucosal pathogens such as influenza viruses and Streptococcus pneumoniae. However, whether systemic immunization induces protective mucosal immunity remains a controversial topic. Here we reviewed the current literature and discussed the potential of systemic routes of immunization for the induction of mucosal immunity.

Background: Study of human coronavirus and other virus-associated respiratory illnesses is needed to describe their clinical effects on chronically ill, older adults., Methods: A prospective study during 2009 to 2013 clinically assessed acute respiratory illnesses soon after onset and 3 to 4 weeks later in patients aged ≥60 years with chronic lung and heart diseases (group 1, 100 subjects) and healthy adults aged 18 to 40 years (group 2, 101 subjects). Respiratory secretions were tested for nucleic acids of a panel of respiratory viruses. An increase in antibody titer was assessed for 4 coronavirus strains., Results: Virus-associated illnesses (29 [39.1%] of 74 illnesses in group 1 and 59 [48.7%] of 121 illnesses in group 2) occurred in all calendar quarters, most commonly in the first and fourth quarters. Coronaviruses (group 1: 14 [18.9%] illnesses; group 2: 26 [21.5%] illnesses) and enteroviruses/rhinoviruses (group 1: 14 [18.9%] illnesses; group 2: 37 [30.6%] illnesses) were most common. Virus co-infections occurred in 10 illnesses. Illnesses with 9 to 11 symptoms were more common in group 1 (17 [23.0%]) than in group 2 (15 [12.4%]) (P < .05). Compared with group 2, more group 1 subjects reported dyspnea, more severe disease of longer duration, and treatment for acute illness with prednisone and antibiotics. Coronavirus-associated illnesses (percent of illnesses, group 1 vs group 2) were characterized by myalgias (21% vs 68%, P < .01), chills (50% vs 52%), dyspnea (71% vs 24%, P < .01), headache (64% vs 72%), malaise (64% vs 84%), cough (86% vs 68%), sputum production (86% vs 60%), sore throat (64% vs 80%), and nasal congestion (93% vs 96%)., Conclusions: Respiratory illnesses were commonly associated with coronaviruses and enteroviruses/rhinoviruses affecting chronically ill, older patients more than healthy, young adults., (Published by Elsevier Inc.)

Multidrug-resistant Acinetobacter baumannii has become an important causative agent of healthcare associated infections. Hospital- and community-acquired pneumonia is the most common clinical manifestation of A. baumannii infection worldwide and is often associated with high mortality. Most experimental vaccine studies to date have evaluated vaccines against systemic A. baumannii infections following systemic immunization. We recently demonstrated that a mouse model of respiratory A. baumannii infection using the strain LAC-4 results in disease progression that is similar to that observed in humans. Here we used this model in conjunction with an inactivated whole cell vaccine to evaluate the feasibility of developing protective mucosal vaccines against respiratory A. baumannii infection and to investigate the potential mechanism of protection of such vaccines. Our results showed that intranasal immunization with formalin-killed whole cells of the LAC-4 strain elicited mucosal and systemic antigen-specific immune responses, and protected mice against lethal intranasal or intraperitoneal challenges. Compared to naïve mice, immunized mice had significantly fewer bacteria in their lungs, and the pathogen was barely detectable in blood and spleens at 24h post challenge, indicating the ability of immunized mice to control extrapulmonary dissemination of the pathogen. Mechanistic studies using gene-deficient mice, neutropenic mice, or passive immunization showed that B cells and neutrophils, but not FcRγ, played crucial roles in the protection against respiratory A. baumannii challenge of intranasally immunized mice whereas passive transfer of hyperimmune sera only prolonged the survival time of challenged mice by 48 h. These results provide immunological insights for the rational design of novel mucosal vaccines to protect against respiratory A. baumannii infection and demonstrate the feasibility to develop such vaccines., (Copyright © 2014 Crown Copyright and Elsevier Inc. Published by Elsevier Ltd.. All rights reserved.)

Acinetobacter baumannii is an important emerging pathogen in health care-acquired infections and is responsible for severe nosocomial and community-acquired pneumonia. Currently available mouse models of A. baumannii pneumonia show poor colonization with little to no extrapulmonary dissemination. Here, we describe a mouse model of A. baumannii pneumonia using a clinical isolate (LAC-4 strain) that reliably reproduces the most relevant features of human pulmonary A. baumannii infection and pathology. Using this model, we have shown that LAC-4 infection induced rapid bacterial replication in the lungs, significant extrapulmonary dissemination, and severe bacteremia by 24 h postintranasal inoculation. Infected mice showed severe bronchopneumonia and dilatation and inflammatory cell infiltration in the perivascular space. More significantly, 100% of C57BL/6 and BALB/c mice succumbed to 10(8) CFU of LAC-4 inoculation within 48 h. When this model was used to assess the efficacy of antimicrobials, all mice treated with imipenem and tigecycline survived a lethal intranasal challenge, with minimal clinical signs and body weight loss. Moreover, intranasal immunization of mice with formalin-fixed LAC-4 protected 40% of mice from a lethal (100× 100% lethal dose) intraperitoneal challenge. Thus, this model offers a reproducible acute course of A. baumannii pneumonia without requiring additional manipulation of host immune status, which will facilitate the development of therapeutic agents and vaccines against A. baumannii pneumonia in humans.

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at pH 2 and 59°C. This extremophile is remarkable by the absence of a cell wall or an S-layer. Treating the cells with Triton X-100 at pH 3 allowed the extraction of all of the cell surface glycoproteins while keeping cells intact. The extracted glycoproteins were partially purified by cation-exchange chromatography, and we identified five glycoproteins by N-terminal sequencing and mass spectrometry of in-gel tryptic digests. These glycoproteins are positive for periodic acid-Schiff staining, have a high content of Asn including a large number in the Asn-X-Ser/Thr sequon and have apparent masses that are 34-48% larger than the masses deduced from their amino acid sequences. The pooled glycoproteins were digested with proteinase K and the purified glycopeptides were analyzed by NMR. Structural determination showed that the carbohydrate part was represented by two structures in nearly equal amounts, differing by the presence of one terminal mannose residue. The larger glycan chain consists of eight residues: six hexoses, one heptose and one sugar with an unusual residue mass of 226 Da which was identified as 6-deoxy-6-C-sulfo-D-galactose (6-C-sulfo-D-fucose). Mass spectrometry analyses of the peptides obtained by trypsin and chymotrypsin digestion confirmed the principal structures to be those determined by NMR and identified 14 glycopeptides derived from the main glycoprotein, Ta0280, all containing the Asn-X-Ser/Thr sequons. Thermoplasma acidophilum appears to have a "general" protein N-glycosylation system that targets a number of cell surface proteins.

Acinetobacter baumannii is an emerging bacterial pathogen that causes nosocomial pneumonia and other infections. Although it is recognized as an increasing threat to immunocompromised patients, the mechanism of host defense against A. baumannii infection remains poorly understood. In this study, we examined the potential role of macrophages in host defense against A. baumannii infection using in vitro macrophage culture and the mouse model of intranasal (i.n.) infection. Large numbers of A. baumannii were taken up by alveolar macrophages in vivo as early as 4 h after i.n. inoculation. By 24 h, the infection induced significant recruitment and activation (enhanced expression of CD80, CD86 and MHC-II) of macrophages into bronchoalveolar spaces. In vitro cell culture studies showed that A. baumannii were phagocytosed by J774A.1 (J774) macrophage-like cells within 10 minutes of co-incubation, and this uptake was microfilament- and microtubule-dependent. Moreover, the viability of phagocytosed bacteria dropped significantly between 24 and 48 h after co-incubation. Infection of J774 cells by A. baumannii resulted in the production of large amounts of proinflammatory cytokines and chemokines, and moderate amounts of nitric oxide (NO). Prior treatment of J774 cells with NO inhibitors significantly suppressed their bactericidal efficacy (P<0.05). Most importantly, in vivo depletion of alveolar macrophages significantly enhanced the susceptibility of mice to i.n. A. baumannii challenge (P<0.01). These results indicate that macrophages may play an important role in early host defense against A. baumannii infection through the efficient phagocytosis and killing of A. baumannii to limit initial pathogen replication and the secretion of proinflammatory cytokines and chemokines for the rapid recruitment of other innate immune cells such as neutrophils.

Allergic asthma is a dysregulation of the immune system which leads to the development of Th2 responses to innocuous antigens (allergens). Some infections and microbial components can re-direct the immune response toward the Th1 response, or induce regulatory T cells to suppress the Th2 response, thereby inhibiting the development of allergic asthma. Since Acinetobacter baumannii infection can modulate lung cellular and cytokine responses, we studied the effect of A. baumannii in modulating airway eosinophilia in a mouse model of allergic asthma. Ovalbumin (OVA)-sensitized mice were treated with live A. baumannii or phosphate buffered saline (PBS), then intranasally challenged with OVA. Compared to PBS, A. baumannii treatment significantly reduced pulmonary Th2 cytokine and chemokine responses to OVA challenge. More importantly, the airway inflammation in A. baumannii-treated mice was strongly suppressed, as seen by the significant reduction of the proportion and the total number of eosinophils in the bronchoalveolar lavage fluid. In addition, A. baumannii-treated mice diminished lung mucus overproduction and pathology. However, A. baumannii treatment did not significantly alter systemic immune responses to OVA. Serum OVA-specific IgE, IgG1 and IgG2a levels were comparable between A. baumannii- and PBS-treated mice, and tracheobronchial lymph node cells from both treatment groups produced similar levels of Th1 and Th2 cytokines in response to in vitro OVA stimulation. Moreover, it appears that TLR-4 and IFN-γ were not directly involved in the A. baumannii-induced suppression of airway eosinophilia. Our results suggest that A. baumannii inhibits allergic airway inflammation by direct suppression of local pulmonary Th2 cytokine responses to the allergen.

Archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) is a safe mucosal adjuvant that elicits long lasting and memory boostable mucosal and systemic immune responses to model antigens such as ovalbumin. In this study, we evaluated the potential of the AMVAD system for eliciting protective immunity against mucosal bacterial infections, using a mouse model of intranasal Francisella tularensis LVS (LVS) challenge. Intranasal immunization of mice with cell free extract of LVS (LVSCE) adjuvanted with the AMVAD system (LVSCE/AMVAD) induced F. tularensis-specific antibody responses in sera and bronchoalveolar lavage fluids, as well as antigen-specific splenocyte proliferation and IL-17 production. More importantly, the AMVAD vaccine partially protected the mice against a lethal intranasal challenge with LVS. Compared to LVSCE immunized and naïve mice, the LVSCE/AMVAD immunized mice showed substantial to significant reduction in pathogen burdens in the lungs and spleens, reduced serum and pulmonary levels of proinflammatory cytokines/chemokines, and longer mean time to death as well as significantly higher survival rates (p<0.05). These results suggest that the AMVAD system is a promising mucosal adjuvant and vaccine delivery technology, and should be explored further for its applications in combating mucosal infectious diseases.

Little is known about the prevalence of mucosal antibodies induced by infection with human coronaviruses (HCoV), including HCoV-229E and -OC43 and recently described strains (HCoV-NL63 and -HKU1). By enzyme-linked immunosorbent assay, we measured anti-HCoV IgG antibodies in serum and IgA antibodies in nasal wash specimens collected at seven U.S. sites from 105 adults aged 50 years and older (mean age, 67 ± 9 years) with chronic obstructive pulmonary disease. Most patients (95 [90%]) had at least one more chronic disease. More patients had serum antibody to each HCoV strain (104 [99%] had antibody to HCoV-229E, 105 [100%] had antibody to HCoV-OC43, 103 [98%] had antibody to HCoV-NL63, and 96 [91%] had antibody to HCoV-HKU1) than had antibody to each HCoV strain in nasal wash specimens (12 [11%] had antibody to HCoV-229E, 22 [22%] had antibody to HCoV-OC43, 8 [8%] had antibody to HCoV-NL63, and 31 [31%] had antibody to HCoV-HKU1), respectively (P < 0.0001). The proportions of subjects with IgA antibodies in nasal wash specimens and the geometric mean IgA antibody titers were statistically higher for HCoV-OC43 and -HKU1 than for HCoV-229E and -NL63. A higher proportion of patients with heart disease than not had IgA antibodies to HCoV-NL63 (6 [16%] versus 2 [3%]; P = 0.014). Correlations were highest for serum antibody titers between group I strains (HCoV-229E and -NL63 [r = 0.443; P < 0.0001]) and between group II strains (HCoV-OC43 and -HKU1 [r = 0.603; P < 0.0001]) and not statistically significant between HCoV-NL63 and -OC43 and between HCoV-NL63 and -HKU1. Patients likely had experienced infections with more than one HCoV strain, and IgG antibodies to these HCoV strains in serum were more likely to be detected than IgA antibodies to these HCoV strains in nasal wash specimens.

Mucosal infections and associated diseases remain a major socio-economic burden to society. Since parenteral immunizations fail to induce efficient protective immunity at mucosal surfaces, mucosal immunization is a logical approach to prevent and treat mucosally-initiated infections. All currently approved human mucosal vaccines are based on attenuated or killed whole pathogen cells but this strategy does pose safety concerns. Therefore, substantial effort is being invested to develop safe and effective mucosal adjuvants and delivery systems for mucosal vaccines. Encouragingly, some of these have progressed to advanced preclinical and clinical studies. This review discusses the promising preclinical research and the potential applications of several novel mucosal adjuvants and delivery systems: an archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) system, 3',5'-cyclic diguanylic acid (c-di-GMP) and detoxified bacterial AB(5) toxins. The potential and challenges in targeting M cells for mucosal vaccination are also discussed.

Archaeal polar lipids are being evaluated as adjuvants/vaccine delivery systems for mucosal vaccines that can provide protection against pathogens that enter the human host via the mucosal surfaces. Archaeosomes, liposomes made from polar lipids extracted from Archaea, with encapsulated antigens elicit strong antigen-specific systemic immune responses upon systemic or intranasal immunization, but fail to generate mucosal immune responses. However, intranasal immunization of mice with the archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) system, obtained by the interaction of archaeosomes/antigens with multivalent cations, induces robust, antigen-specific IgA responses in nasal and vaginal mucosa, feces, bile, and serum. In addition, strong antigen-specific systemic antibody (serum IgG, IgG(1) and IgG(2a)) and cell-mediated responses, including CD8(+) cytotoxic T lymphocyte, are generated. The responses are sustained over time and are subject to good memory-boost responses. The AMVAD formulations are stable during storage, have a good safety profile and show protective efficacy in a murine model of infection/challenge.

Calcium, a component of approved human vaccines administered via systemic routes, has a good safety profile. Recently, intranasally administered vaccines containing calcium have shown promise in generating mucosal immune responses in animal models. However, the safety of intranasally administered calcium is unknown. This study evaluates the safety of intranasally administered calcium at 2- to 13-fold higher doses than used in experimental vaccines. At a calcium dose of 22 mg/kg, 80% of the Balb/c and 20% of the C57BL/6 mice die within the first 24 hours. At 11.0 mg/kg, there is no overt toxicity in either strain, based on body weight, clinical scores, blood chemistry, and histopathology of major organs at 7 days post administration. In C57BL/6 mice, apart from acute and subacute inflammation in the lungs at up to 3 days post administration, especially at the 22-mg/kg dose, there is no overt toxicity. Doses of calcium up to 11 mg/kg appear to be safe in a mouse model.

3',5'-Cyclic diguanylic acid (cdiGMP) is emerging as a universal bacterial second messenger in regulating bacterial growth on surfaces. It has been recently shown that cdiGMP stimulates innate immunity and enhances antigen-specific humoral and cellular immune responses. We herein report that intranasal (i.n.) administration with cdiGMP induces an acute but transient inflammatory response and activation of dendritic cells in the lungs. Moreover, i.n. immunization of mice with pneumococcal surface adhesion A (PsaA) in conjunction with cdiGMP elicited strong antigen-specific serum immunoglobulin G (IgG) and secretory IgA antibody responses at multiple mucosal surfaces. More importantly, the immunized mice showed significantly reduced nasopharyngeal Streptococcus pneumoniae colonization. These results, for the first time, provide direct evidence for the induction of protection against mucosal bacterial infections by cdiGMP as an adjuvant.

The safety profile of a recently described novel archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) system capable of eliciting robust antigen-specific mucosal and systemic immune responses was evaluated in female Balb/c mice (10/group) using ovalbumin (OVA) antigen. Mice were intranasally immunized (0, 7, and 21 days) with a vaccine comprising 1 microg OVA (0.05 mg/kg body weight) formulated in 0.04 mg total polar lipids extract (2.17 mg/kg body weight) of Methanobrevibacter smithii constituting the AMVAD system. Control groups were similarly immunized with 10-fold higher AMVAD vaccine dose (0.54 mg OVA and 21.7 mg lipid per kg), saline, 10 microg OVA in saline, or 0.04 or 0.4 mg lipid constituting empty AMVAD (no OVA) in saline, or were naive mice. Clinical signs, rectal temperature, and body weight were monitored once daily or as appropriate. Half the mice in each group were euthanized at 2 days after the first immunization. Blood was collected for clinical chemistry analyses. Major organs (heart, lungs, kidneys, liver, spleen, thymus, and brain) were examined macroscopically and histologically. The remaining mice were euthanized at 29 days and blood and organs collected for analyses as done at 2 days. Feces collected at 27 days, and sera, bile, and nasal lavage at 29 days, were assayed for antibody responses. Based on clinical symptoms, temperature, body weight changes, serum clinical chemistry, and tissue histopathology, there were no overt toxicities associated with OVA/AMVAD or empty AMVAD vaccines. There were no antibodies elicited against the lipids comprising the AMVAD system. These results demonstrate that at 10-fold excess dose of that required for vaccine efficacy, intranasally administered AMVAD vaccine appears to be relatively safe.

Background: It has been suggested that exposure to certain microbes and their products, particularly during neonatal and early childhood periods, may shift the immune response towards a T-helper cell (Th) 1 phenotype and thereby prevent the development of and/or alleviate the clinical symptoms of allergic airway diseases., Objective: We evaluated the ability of the live vaccine strain (LVS) of Francisella tularensis to suppress airway eosinophilia and pulmonary pathology in a murine model of allergic airway disease., Methods: C57BL/6 mice were sensitized by intraperitoneal injection of ovalbumin (OVA) on days 1 and 14, and challenged intranasally (i.n.) with OVA on day 21 or thereafter. Some sensitized mice were i.n. treated with live LVS or its cell-free sonicate extract (CFSE) before i.n. OVA challenge. Bronchoalveolar lavage fluid, regional lymph node cells, lung tissues and serum samples were collected 3-7 days after the i.n. challenge., Results: Intranasal and, to a lesser degree, intradermal immunization of OVA-sensitized mice with LVS suppressed the development of airway eosinophilia and associated pulmonary pathology induced by i.n. OVA challenge. Moreover, CFSE prepared from LVS showed a similar inhibitory effect whereas neither LPS nor DNA purified from F. tularensis LVS had such an effect. The inhibition was associated with the reduction in mRNA expression and protein levels of Th2 cytokines IL-5 and IL-13 in the lungs and the enhanced production of OVA-induced IFN-gamma by local draining lymph node cells, but not with the serum levels of OVA-specific IgG1 or IgE., Conclusion: F. tularensis LVS is capable of suppressing allergic airway inflammation probably through a Th1-mediated suppression of an ongoing Th2 response mechanism, and raises the possibility of exploring LVS and its components as potential therapeutic modalities for human allergic asthma.

Intranasal administration of ovalbumin (OVA) formulated in an archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) system prepared by the addition of CaCl2 to small unilamellar archaeosomes (liposomes made from archaeal polar lipids) containing encapsulated OVA, was recently shown to elicit strong and sustained OVA-specific mucosal and systemic immune responses. In this study, we show that the centrifugation/washing and antigen quantization steps required in the standard protocol for obtaining OVA/AMVAD model vaccine formulations can be eliminated by using simpler protocols such as admixing OVA with preformed empty archaeosomes, or by changing the starting ratio (w/w) of archaeal lipid to antigen at the archaeosome preparation stage, prior to the addition of CaCl2 to convert to the AMVAD structures. Irrespective of the vaccine preparation protocol, the AMVAD particle typically comprised of larger spherical structures that had aggregated like a bunch of grapes, and it contained aqueous compartment(s). The anti-OVA IgA antibody responses in vaginal wash, nasal wash, serum, and bile samples, and the anti-OVA IgG antibody responses in sera, in mice intranasally immunized with the OVA/AMVAD formulations prepared by the simplified or the standard protocols, were comparable.

The utility of archaeal polar lipids as an adjuvant/delivery system for elicitation of antigen-specific mucosal immune responses in intranasally administered vaccines was investigated. Although unilamellar archaeosomes (liposomes made from archaeal polar lipids) with encapsulated ovalbumin (OVA/archaeosomes) induced anti-OVA IgG antibody responses in sera, they failed to induce anti-OVA IgA antibody responses at mucosal sites. However, the addition of CaCl2 to convert OVA/archaeosomes into an archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) vaccine (OVA/AMVAD) consisting of larger, particulate, aggregated structures resulted in an efficacious intranasal (i.n.) vaccine. Intranasal immunization of mice with OVA/AMVAD vaccines prepared from various archaeal polar lipid compositions elicited anti-OVA IgA antibody responses in sera, feces, bile, vaginal and nasal wash samples. The i.n. immunization also induced anti-OVA IgG, IgG1 and IgG2a antibody responses in sera, as well as cytotoxic T lymphocyte responses. The mucosal and systemic immune responses induced by OVA/AMVAD immunization were generally sustained over several months, and were subject to memory boost responses. Thus, polar archaeal lipids appear to be promising for developing a non-replicating mucosal adjuvant and vaccine delivery system.

Effects of human immunodeficiency virus type 1 (HIV-1) recombinant envelope glycoprotein vaccines on cell-mediated immune (CMI) responses were assessed in HIV-1-infected patients. Asymptomatic, antiretroviral-treatment-naïve, HIV-1-infected patients with CD4(+) T-cell counts greater than 400/microl received multiple intramuscular injections of HIV-1 IIIB recombinant envelope glycoprotein (rgp160) vaccine or HIV-1 MN recombinant envelope glycoprotein (rgp120) vaccine (eight patients, referred to as the HIV-1 vaccinees) or placebo or hepatitis B vaccine (three patients, referred to as the controls). Lymphocyte proliferation in response to HIV-1 envelope glycoproteins, both homologous and heterologous to the HIV-1 immunogens, was absent prior to study treatment in all patients but increased significantly during the vaccination series and after the final vaccination in HIV-1 vaccinees (P < 0.05) and remained absent in control patients. In flow cytometric analyses of intracellular cytokines, T-cell receptor stimulation with an anti-CD3 antibody induced gamma interferon (IFN-gamma) expression by activated CD4(+) and CD8(+) lymphocytes at greater frequencies than did stimulation with recombinant envelope glycoprotein and p24 of HIV-1 (P < 0.05). Mean frequencies of HIV-1 envelope glycoprotein-stimulated, activated intra-cellular IFN-gamma-producing CD4(+) and CD8(+) lymphocytes and of interleukin-2-producing CD4(+) lymphocytes did not increase after vaccination, but cytokine-producing cells were detectable in some patients. Comparing pre- to post-HIV-1 vaccination time points, changes in frequencies of activated, IFN-gamma-producing CD4(+) cells correlated inversely with changes in lymphocyte proliferation in response to recombinant envelope glycoprotein in HIV-1 vaccinees (P < 0.05). Increased CMI responses to HIV-1 envelope glycoprotein measured by lymphocyte proliferation were associated with HIV-1 recombinant envelope glycoprotein vaccines.

Archaeosomes are liposomes made from the polar ether lipids of Archaea. These lipids are unique and distinct in structure from the ester lipids found in Eukarya and Bacteria. The regularly branched and usually fully saturated isopranoid chains of archaeal polar lipids are attached via ether bonds to the sn-2,3 carbons of the glycerol backbone(s). The polar head groups are usually the same as those encountered in the ester lipids from the other two domains, except that phosphatidylcholine is rarely present. These lipid structures provide formulary advantages, and contribute to the excellent physico-chemical stability of the archaeosomes and their efficacy as self-adjuvanting vaccine delivery vesicles. The uptake of archaeosomes by phagocytic cells is several folds greater than that of liposomes made from ester lipids. In addition, archaeosomes enhance the recruitment and activation of professional antigen presenting cells in vivo, and deliver the antigen to both MHC class I and II pathways for antigen presentation, without eliciting overt inflammatory responses. In murine models, systemic administration of archaeosomes containing encapsulated antigen(s) elicits strong and sustained antigen-specific antibody responses which are comparable, in some formulations, to those obtained with Freund's adjuvant. Additionally, archaeosomes promote robust antigen-specific cell-mediated immunity, including CD8+ CTL responses. The immune responses induced by archaeosomes are sustained over long periods and exhibit strong memory responses. More importantly, immunization of mice with archaeosome-based vaccines induces robust protective immunity against intracellular pathogens, and prophylactic and therapeutic efficacies against the development of experimental cancers. Extensive murine model studies suggest that archaeosomes are safe.

Binding of antibodies to oligomeric envelope glycoprotein of R5-tropic primary isolates of human immunodeficiency virus type 1 (HIV-1) was studied by flow cytometry using sera from HIV-1 vaccine recipients and clade B and C HIV-1-infected patients, and monoclonal and polyclonal antibodies to neutralizing epitopes of HIV-1. Vaccine recipients received recombinant canarypox virus vaccine expressing HIV-1 gene products, and SF-2 recombinant gp120 subunit vaccine. Anti-gp120 neutralizing antibodies including human monoclonal antibody 2G12 and goat polyclonal anti-serum to V3 loop peptide [peptide T1-SP10MN(A)] bound to HIV-1-infected cells. Sera from vaccine recipients bound to HIV-1-infected cells, but at levels lower than did infected patient sera.

The present study evaluated the potential of archaesomes, prepared from the total polar lipids extracted from Methanobrevibacter smithii, as adjuvants for combination (multivalent) vaccines. Groups of Balb/c mice were immunized subcutaneously at day 0 and 21 with one of the following vaccines: trivalent vaccine formulated by the simultaneous co-encapsulation of bovine serum albumine (BSA), ovalbumin (OVA) and hen egg lysozyme (HEL) into archaeosomes (CEC vaccine); an univalent archaeosome vaccine (UVE vaccine) containing either BSA, OVA or HEL; or an admixture vaccine (AMC vaccine) consisting of the three UVE vaccines. Serum specific antibody (IgG + M) responses were determined at day 32, 112 and 203, and specific IgG1 and IgG2a responses were determined at day 112. Mice immunized with the CEC of AMC vaccine developed strong and sustained specific antibody responses to all three antigens at a magnitude similar to those seen in control mice immunized with UVE vaccines. Moreover, the serum BSA-, OVA-, and HEL-specific IgG1 and IgG2a levels in the CEC and AMC immunized mice were overall comparable to those of the UVE immunized control mice. Boosting CEC and AMC vaccinated mice with antigens alone at day 203 elicited strong antibody memory responses, comparable to those in the UVE vaccinated groups. These results show that archaeosomes could be used as adjuvants in developing combination vaccines.

Archaeosomes prepared from total polar lipids extracted from six archaeal species with divergent lipid compositions had the capacity to deliver antigen for presentation via both MHC class I and class II pathways. Lipid extracts from Halobacterium halobium and from Halococcus morrhuae strains 14039 and 16008 contained archaetidylglycerol methylphosphate and sulfated glycolipids rich in mannose residues, and lacked archaetidylserine, whereas the opposite was found in Methanobrevibacter smithii, Methanosarcina mazei and Methanococcus jannaschii. Annexin V labeling revealed a surface orientation of phosphoserine head groups in M. smithii, M. mazei and M. jannaschii archaeosomes. Uptake of rhodamine-labeled M. smithii or M. jannaschii archaeosomes by murine peritoneal macrophages was inhibited by unlabeled liposomes containing phosphatidylserine, by the sulfhydryl inhibitor N-ethylmaleimide, and by ATP depletion using azide plus fluoride, but not by H. halobium archaeosomes. In contrast, N-ethylmaleimide failed to inhibit uptake of the four other rhodamine-labeled archaeosome types, and azide plus fluoride did not inhibit uptake of H. halobium or H. morrhuae archaeosomes. These results suggest endocytosis of archaeosomes rich in surface-exposed phosphoserine head groups via a phosphatidylserine receptor, and energy-independent surface adsorption of certain other archaeosome composition classes. Lipid composition affected not only the endocytic mechanism, but also served to differentially modulate the activation of dendritic cells. The induction of IL-12 secretion from dendritic cells exposed to H. morrhuae 14039 archaeosomes was striking compared with cells exposed to archaeosomes from 16008. Thus, archaeosome types uniquely modulate antigen delivery and dendritic cell activation.

Archaeosome adjuvants formulated as archaeal ether glycerolipid vesicles induce strong CD4(+) as well as CD8(+) CTL responses to entrapped soluble antigens. Immunization of mice with ovalbumin (OVA) entrapped in archaeosomes composed of the total polar lipids of Methanobrevibacter smithii resulted in a potent OVA-specific CD8(+) T-cell response, and subsequently, the mice dramatically resisted solid tumor growth of OVA-expressing EG.7 cells and lung metastasis of B16OVA melanoma cells. Prophylactic protection was antigen-specific because tumor curtailment was not seen in mice injected with antigen-free archaeosomes. Similarly, there was no protection against B16 melanoma cells lacking OVA expression. Furthermore, in vivo depletion of CD8(+) T cells abrogated the protective response, indicating that the antitumor immunity was mediated by CTLs. Depletion of CD4(+) T cells also resulted in partial loss of tumor protection, suggesting a beneficial role for T-helper cells. Interestingly OVA-archaeosomes induced enhanced CTL response in the absence of interleukin 12 and IFN-gamma. Furthermore, interleukin 12-deficient mice mounted strong tumor protection. However, IFN-gamma-deficient mice, despite the strong CTL response, were only transiently protected, revealing a need for IFN-gamma response in tumor protection. Archaeosomes also facilitated therapeutic protection when injected into mice concurrent with tumor cells. Interestingly, even archaeosomes lacking entrapped antigen mediated therapeutic protection, and this correlated to the activation of innate immunity as evident by the increased tumor-infiltrating natural killer and dendritic cells. Thus, archaeosomes represent effective tumor antigen delivery vehicles that can mediate protection by activating both innate as well as acquired immunity.

Archaeosomes, liposomes made from polar ether lipids of archaea, show promise for vaccine and drug delivery applications. The potential toxicity of intravenously (14, 70, or 140 mg/kg/day for 5 consecutive days) and orally (gavaged at 55, 275, or 550 mg/kg/day for 10 consecutive days) administered unilamellar archaeosomes, prepared from the total polar lipids (TPLs) extracted from several species of archaea, was assessed in female BALB/c mice. Liposomes prepared from an ester phospholipid composition were included for comparative purposes. Control groups of mice were administered 0.1 ml phosphate-buffered saline (PBS) by either route. Animals were monitored at least once daily for temperature, body weight, and clinical signs of adverse reactions. One day after the last dose, the mice were sacrificed. Blood was collected for selected biochemical/enzyme analyses, and the major organs (heart, lungs, liver, spleen, kidneys) were weighed and examined macroscopically. In addition, the spleens were examined histologically. At the two lower dosages of intravenously administered vesicles, there were no significant indications of toxicity, as compared with the PBS-administered control group. At the highest intravenous dose of 140 mg/kg/day, archaeosomes prepared from the TPL of the extreme halophiles, Halobacterium salinarum and Natronobacterium magadii, indicated potential toxicity, as evidenced by clinical signs (hyperactivity and/or piloerection), drop in body temperature, and loss in body weight. Spleens from mice administered some archaeosomes types, primarily at the highest intravenous dose tested, were enlarged, had increased organ weight, and microscopic examination revealed mild to moderate expansion of the red pulp with increased numbers of hematopoietic cells, but no changes in the white pulp. There were similar clinical signs at one or more of the higher oral doses of the ester liposomes and some of the archaeosome types; however, no other apparent toxicity was observed. Based on this limited mouse study, archaeosomes were generally well tolerated after intravenous or oral delivery at the dosages so indicated in this study.

Archaeosomes, liposomes prepared from the polar ether lipids extracted from Archaea, demonstrate great potential as immunomodulating carriers of soluble antigens, promoting humoral and cell mediated immunity in the vaccinated host. The safety of unilamellar archaeosomes prepared from the total polar lipids (TPL) of Halobacterium salinarum, Methanobrevibacter smithii or Thermoplasma acidophilum was evaluated in female BALB/c mice using ovalbumin (OVA) as the model antigen. Groups of 6-8 mice were injected (0.1 mL final volume) subcutaneously at 0 and 21 days, with phosphate buffered saline (PBS), 11 microg OVA in PBS, 1.25 mg of antigen-free archaeosomes in PBS (ca 70 mg/kg body wt), or PBS containing 11-20 microg OVA encapsulated in 1.25mg archaeosomes. Animals were monitored daily for injection site reactions, body weight,temperature and clinical signs of adverse reactions. Sera were collected on days 1, 2, 22, and 39 for analyses of creatine phosphokinase. Mice were sacrificed on 39 d, sera were collected for biochemical analyses, and major organs (liver, spleen, kidneys, heart, lungs) were weighed and examined macroscopically. There were no indications of adverse reactions or toxicity associated with any of the archaeosome adjuvants. None of the antigen-free archaeosomes elicited significant anti lipid antibodies when subcutaneously injected (1 mg each at 0, 1, 2, and 4 weeks) in mice, although anti H. salinarum lipid antibodies were detected. These antilipid antibodies cross-reacted with the TPL of T. acidophilum archaeosomes but not with the TPL of M. smithii archaeosomes nor with lipids of ester liposomes made from L-alpha-dimyristoylphosphatidylcholine (DMPC), L-alpha-dimyristoylphosphatidylglycerol (DMPG), and cholesterol (CHOL). In vitro hemolysis assay on mouse erythrocytes indicated no lysis with M. smithii or T. acidophilum archaeosomes at up to 2.5 mg/mL concentration. At this concentration, H. salinarum archaeosomes and DMPC/DMPG/CHOL ester liposomes caused about 2% and 4% hemolysis, respectively. Based on this mouse model evaluation, archaeosomes are well-tolerated and appear relatively safe for potential vaccine applications.

T cell memory to human immunodeficiency virus type 1 (HIV-1) antigens and anti-HIV-1 cytotoxic T lymphocyte (CTL) activity were assessed after administration of live canarypox virus (ALVAC) expressing HIV-1 env, gag, and protease (vCP205) vaccine given alone, vCP205 given with SF-2 recombinant gp120 (rgp120) vaccine, and placebos at 0, 1, 3, and 6 months. Healthy, HIV-1-uninfected subjects reporting high-risk and low-risk behavior for HIV-1 were enrolled. Anti-HIV-1 Env CD8(+) CTLs (HIV-1(MN) and/or HIV-1 subtype B and C primary isolate sequences) were detected in 12 (60%) and anti-HIV-1 Gag CD8(+) CTLs in 7 (35%) of the 20 vCP205 vaccine recipients tested by CTL assay 3.5 months after the final immunization. Fourteen days after the fourth immunization, lymphocyte proliferation in response to HIV-1 Gag antigen was detected in 14 (48%) of 29 vCP205 vaccine recipients, but secreted cytokine levels to HIV-1 Gag antigen were not above unstimulated levels. Coadministration of SF-2 rgp120 vaccine with vCP205 vaccine enhanced lymphocyte proliferation in response to HIV-1 envelope glycoprotein and broadened the envelope-stimulated cytokine secretion pattern, so that it consisted of both Th1 and Th2 cytokines compared with only interferon gamma (IFN-gamma) after vCP205 vaccine given alone. There was a possible association between HIV-1 envelope glycoprotein-stimulated interleukin 2 secretion and CD8(+) CTLs against HIV-1 envelope glycoprotein, and an inverse relation between lymphocyte proliferation and CTLs against HIV-1 Gag antigens. Thus, a durable anti-HIV-1 CD8(+) CTL response was detected after immunization with the live canarypox virus vaccine and preexisting helper T cell memory responses did not necessarily predict later CD8(+) CTL activity.

Protective immunity to intracellular bacterial pathogens usually requires the participation of specific CD8+ T cells. Natural exposure of the host to sublethal infection, or vaccination with attenuated live vaccines are the most effective means of eliciting prolonged protective cell-mediated immunity against this class of pathogens. The ability to replace these immunization strategies with defined sub-unit vaccines would represent a major advance for clinical vaccinology. The present study examines the ability of novel liposomes, termed archaeosomes, made from the polar lipids of various Archaeobacteria to act as self-adjuvanting vaccine delivery vehicles for such defined acellular antigens. Using infection of mice with Listeria monocytogenes as a model system, this study clearly demonstrates the ability of defined, archaeosome-entrapped antigens to elicit rapid and prolonged specific immunity against a prototypical intracellular pathogen. In this regard, all of the tested archaeosomes were superior to conventional liposomes.