Masking terminal neo-epitopes of linear peptides through glycosylation favours immune responses towards core epitopes producing parental protein bound antibodies.

Glycosylation of hydrophobic peptides at one terminus effectively increases their water-solubility, and conjugation through the opposing end to a carrier protein, renders them more immunogenic. Moreover, the glycosylation minimizes antibody responses to potentially deleterious, non-productive terminal neo-epitope regions of the peptides, and consequently shifts peptide immunogenicity towards the core amino acid residues. As proof of concept, glycopeptide-protein conjugates related to influenza hemagglutinin (HA), neuraminidase (NA), and the dimerization loop region of human epidermal growth factor receptor 2 (Her2), demonstrated a favorable production of core peptide specific antibodies as determined by ELISA studies. Furthermore, glycosylated Her2 peptide conjugate antisera were also shown to recognize full length Her2 protein by ELISA and at the cell surface through flow cytometry analysis. In contrast, unmasked peptide conjugates generated significant antibody populations that were specific to the terminal neo-epitope of the peptide immunogen that are notably absent in parental proteins. Antibodies generated in this manner to peptides in the dimerization loop of Her2 are also functional as demonstrated by the growth inhibition of Her2 expressing SKBR3 carcinoma cells. This method provides a technique to tailor-make epitope-specific antibodies that may facilitate vaccine, therapeutic and diagnostic antibody development.

Safety evaluation of calcium administered intranasally to mice.

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.

Archaeal glycolipid adjuvanted vaccines induce strong influenza-specific immune responses through direct immunization in young and aged mice or through passive maternal immunization.

Vaccine induced responses are often weaker in those individuals most susceptible to infection, namely the very young and the elderly, highlighting the need for safe and effective vaccine adjuvants. Herein we evaluated different archaeosome formulations as an adjuvant to the H1N1 influenza hemagglutinin protein and compared immune responses (anti-HA IgG and hemagglutination inhibition assay titers) as well as protection to an influenza A virus (strainA/PuertoRico/8/1934H1N1)homologous challenge to those generated using a squalene-based oil-in-water nano-emulsion, AddaVax™ in a murine model. The impact of age (young adult vs aged) on vaccine induced immune responses as well as the protection in pups due to the transfer of maternal antibodies was measured. Overall, we show that archaeal lipid based adjuvants can induce potent anti-HA responses in young and aged mice that can also be passed from vaccinated mothers to pups. Furthermore, young and aged mice immunized with archaeal lipid adjuvants as well as pups from immunized mothers were protected from challenge with influenza. In addition, we show that a simple admixed archaeosome formulation composed of a single sulfated glycolipid namely sulfated lactosylarchaeol (SLA; 6'-sulfate-ß-D-Galp-(1,4)-ß-D-Glcp-(1,1)-archaeol) can give equal or better protection compared to AddaVax™ or the traditional antigen-encapsulated archaeosome formulations.

Evaluation of two yellow fever vaccines for routine immunization programs in Argentina.

Although highly effective vaccines have been available for almost 70 years, an estimated 200,000 cases of YF, including 30,000 deaths, still occur annually. This study evaluated the safety of two yellow fever (YF) vaccines [Stamaril and Vacina Contra Febre Amarela (VCFA)]. A total of 2,514 subjects were randomized equally to receive Stamaril or VCFA. Immediate reactions occurring within 30 minutes after vaccination, and solicited local and systemic reactions occurring within eight days, were monitored. Unsolicited local, systemic adverse events and serious adverse events (SAE) were recorded for 21 days after vaccination. Solicited local and systemic adverse reactions were reported by 15.3-17.6% and 30.4-31.6% of the Stamaril and VCFA groups, respectively. Only 56 of the 2,514 study subjects (2.2%) reported a severe solicited adverse reaction, 25 in the Stamaril group (1.99%) and 31 in the VFCA group (2.49%), (p=0.403). Ten subjects (0.8%) in each group reported at least one severe solicited local reaction (p = 0.988). A total of 18 Stamaril subjects (1.43%) and 21 VCFA subjects (1.68%) reported at least one severe solicited systemic reaction (p = 0.617) One SAE considered related to vaccination occurred, polymyalgia in the VCFA group. No immediate reactions to vaccination were seen. Vaccine-related unsolicited events were infrequent, 1.4% in the Stamaril group and 2.0% VCFA group, generally of mild or moderate intensity. We conclude that the safety profiles of Stamaril and VCFA support routine vaccination to prevent YF in residents of and travelers to endemic areas of South America and Africa.

Structural characterization of archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) formulations prepared by different protocols and their efficacy upon intranasal immunization of mice.

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.

Safety of intranasally administered archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) vaccine in mice.

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.

Neuroendocrine profiling of humans receiving cardiac allografts.

BACKGROUND: Several studies have investigated changes in circulating hormones and markers of cardiac status after heart transplantation in humans. As a result, plasma levels of various hormones and autocoids have been associated with cardiac allograft rejection status. However, no clear associations can be defined given the highly contradictory nature of the available literature. METHODS: In this study of 69 consecutive heart transplant patients followed for >2 years we examine the relationship between neurohumors potentially related to allograft rejection and endomyocardial biopsy grade of rejection (according to the ISHLT) and hemodynamic status. Markers assessed include brain natriuretic peptide (BNP), amino-terminal pro-BNP (N-BNP), atrial natriuretic factor (ANF), adrenomedullin, interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, troponin C and C-reactive protein. RESULTS: The highest plasma levels for most neurohumors were found shortly after surgery and showed a trend towards normalization with time. BNP and N-BNP were the only significantly elevated plasma analytes for patients with Grade 3 rejection as compared with other ISHLT grades. ANF plasma levels correlated with BNP and N-BNP in Grades 0 to 2, but not in Grade 3, suggesting that in this rejection grade the usual coordinated changes observed in BNP and ANF secretion no longer exist. Cardiac filling pressures were correlated with plasma BNP, N-BNP and ANF levels only for Grades 0 and 1. CONCLUSIONS: The timing of blood sampling after transplantation influences the level of the neurohumors measured, which may help explain the conflicting literature reports on the association between neurohumor levels and rejection grade. The significant increase in circulating levels of BNP and N-BNP observed in most cases of Grade 3 rejection occurred with no apparent relationship to post-transplantation time, which suggests a specific influence of acute rejection on BNP gene expression.

Participation of G proteins in natriuretic peptide hormone secretion from heart atria.

The involvement of G proteins in the mechanism underlying the increased atrial natriuretic factor (ANF) secretion observed after atrial muscle stretch (stretch-secretion coupling) was assessed using a combined pharmacological, immunocytochemical, and tissue fractionation approach. It was found that G(i/o) inhibition by pertussis toxin (PTX) abolished stretch-secretion coupling without affecting baseline secretion through a mechanism that is independent of G(q) signaling agonists. Mastoparan-7, a G(i/o) agonist, significantly increased ANF secretion even in the absence of muscle stretch through a PTX-sensitive mechanism. By confocal and electron immunocytochemistry, ANF and G(o) partially colocalized, whereas ultracentrifugation analysis suggested the presence of two populations of granules, one of which was partially associated with G(o), as demonstrated by Western blotting. PTX did not affect basal or endothelin-1-stimulated ANF secretion, in line with the view that endothelin-1 signals mainly through G(q). It is concluded there are at least two types of regulated secretory processes in atrial cardiocytes: one is acutely responsive to muscle stretch and is PTX sensitive, and the other is G(q)mediated and PTX insensitive and may be responsible for changes in secretion after chronic changes in the neuroendocrine environment.

Intranasal immunization with an archaeal lipid mucosal vaccine adjuvant and delivery formulation protects against a respiratory pathogen challenge.

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.

Mucosal and systemic immune responses by intranasal immunization using archaeal lipid-adjuvanted vaccines.

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.

Efecto antioxidante y citoprotector del Solanum tuberosum (papa) en la mucosa gástrica de animales de experimentación / Antioxidant and cytoprotection effects of Solanum tuberosum (potato) on gastric mucosa in experimental animals

Introducción: Existen alternativas terapéuticas con productos naturales oriundos usados de manera empírica en la población. Tal es el caso del zumo de papa (Solanum tuberosum) usado para problemas de mucosa gástrica. Objetivos: Evaluar la capacidad antioxidante y el efecto citoprotector a la mucosa gástrica del zumo de papa (Solanum tuberosum). Diseño: Experimental. Institución: Centro de Investigación de Bioquímica y Nutrición de la Facultad de Medicina, Universidad Nacional Mayor de San Marcos, Lima. Materiales biológicos: Solanum tuberosum, variedad Tomasa y ratas albinas machos. Métodos: Se administró vía oral post ayuno las fracciones de sobrenadante y sedimento del zumo de Solanum tuberosum. Una hora después se administró alcohol como injuria de mucosa gástrica. Por laparotomía abdominal se obtuvo el tejido gástrico. Se midió en la mucosa gástrica el estrés oxidativo por lipoperoxidación, la formación de moco por alcian blue y la protección midiendo la extensión del área lacerada en imagen digitalizada. Principales medidas de resultados: Capacidad antioxidante y efecto citoprotector a la mucosa gástrica. Resultados: El sobrenadante de la dosis 5 mL/ kg produjo mayor protección al estrés oxidativo; el precipitado en dosis 5 mL/kg presentó mayor producción de moco, sin superar al control. El precipitado 20 mL/kg produjo mayor citoprotección (73,8 por ciento). Conclusión: La fracción sobrenadante del zumo de la papa (Solanum tuberosum) posee actividad de defensa antioxidante y la fracción del sedimento, mayor actividad citoprotectora de la mucosa gástrica.

Background: There are therapeutic alternatives in Peru with empirical native products such as potato juice. Objectives: To assess the antioxidant capacity and the gastric mucosa cytoprotection effect by Solanum tuberosum juice. Design: Experimental. Institution: Research Center of Biochemistry and Nutrition, Faculty of Medicine, Universidad Nacional Mayor de San Marcos. Biological materials: Solanum tuberosum, tomasa variety, and male albino rats. Methods: Oral solid fractions of supernatant and sediment from Solanum tuberosum juice were administered to fasting rats and an hour later, alcohol was given for gastric injury; gastric tissue was obtained by laparotomy. Oxidative stress was measured in the gastric mucosa by lipoperoxidation, formation of mucus by alcian blue, and the extent of protection by measuring lacerated areas in scanned image. Main outcome measures: Antioxidant capacity and gastric mucosa cytoprotection. Results: The 5 mL/kg supernatant produced greater oxidative stress protection; the 5 mL/kg pellet dose showed increased production of mucus, without exceeding controls. The 20 mL/kg precipitate produced greater cytoprotection, 73,8 per cent. Conclusion: Supernatant fraction of Solanum tuberosum juice has antioxidant effect and the fraction of the sediment increased cytoprotective activity on gastric mucosa.