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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.

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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.

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.

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.