pH responsiveness of fibrous assemblies of repeat-sequence amphipathic α-helix polypeptides.

We reported previously that our designed polypeptide α3 (21 residues), which has three repeats of a seven-amino-acid sequence (LETLAKA)3, forms not only an amphipathic α-helix structure but also long fibrous assemblies in aqueous solution. To address the relationship between the electrical states of the polypeptide and its α-helix and fibrous assembly formation, we characterized mutated polypeptides in which charged amino acid residues of α3 were replaced with Ser. We prepared the following polypeptides: 2Sα3 (LSTLAKA)3, in which all Glu residues were replaced with Ser residues; 6Sα3 (LETLASA)3, in which all Lys residues were replaced with Ser; and 2S6Sα3 (LSTLASA)3; in which all Glu and Lys residues were replaced with Ser. In 0.1M KCl, 2Sα3 formed an α-helix under basic conditions and 6Sα3 formed an α-helix under acid conditions. In 1M KCl, they both formed α-helices under a wide pH range. In addition, 2Sα3 and 6Sα3 formed fibrous assemblies under the same buffer conditions in which they formed α-helices. α-Helix and fibrous assembly formation by these polypeptides was reversible in a pH-dependent manner. In contrast, 2S6Sα3 formed an α-helix under basic conditions in 1M KCl. Taken together, these findings reveal that the charge states of the charged amino acid residues and the charge state of the Leu residue located at the terminus play an important role in α-helix formation.

Role of positions e and g in the fibrous assembly formation of an amphipathic α-helix-forming polypeptide.

We previously characterized α3, a polypeptide that has a three times repeated sequence of seven amino acids (abcdefg: LETLAKA) and forms fibrous assemblies composed of amphipathic α-helices. Upon comparison of the amino acid sequences of α3 with other α-helix forming polypeptides, we proposed that the fibrous assemblies were formed due to the alanine (Ala) residues at positions e and g. Here, we characterized seven α3 analog polypeptides with serine (Ser), glycine (Gly), or charged residues substituted for Ala at positions e and g. The α-helix forming abilities of the substituted polypeptides were less than that of α3. The polypeptides with amino acid substitutions at position g and the polypeptide KEα3, in which Ala was substituted with charged amino acids, formed few fibrous assemblies. In contrast, polypeptides with Ala replaced by Ser at position e formed ß-sheets under several conditions. These results show that Ala residues at position e and particularly at position g are involved in the formation of fibrous assemblies.

Intranasal influenza vaccination using a new synthetic mucosal adjuvant SF-10: induction of potent local and systemic immunity with balanced Th1 and Th2 responses.

BACKGROUND: We found previously that bovine pulmonary Surfacten® used in newborns with acute respiratory distress syndrome is a safe and efficacious antigen vehicle for intranasal vaccination. OBJECTIVES: The objective of this study was to industrially produce a synthetic adjuvant mimicking Surfacten® for clinical use without risk of bovine spongiform encephalopathy. METHODS: We selected three Surfacten lipids and surfactant protein (SP)-C as essential constituents for adjuvanticity. For replacement of the hydrophobic SP-C, we synthesized SP-related peptides and analyzed their adjuvanticity. We evaluated lyophilization to replace sonication for the binding of influenza virus hemagglutinin (HA) to the synthetic adjuvant. We also added a carboxy vinyl polymer (CVP) to the synthetic adjuvant and named the mixture as SF-10 adjuvant. HA combined with SF-10 was administered intranasally to mice, and induction of nasal-wash HA-specific secretory IgA (s-IgA) and serum IgG with Th1-/Th2-type cytokine responses in nasal cavity and virus challenge test were assessed. RESULTS AND CONCLUSIONS: Intranasal immunization with HA-SF-10 induced significantly higher levels of anti-HA-specific nasal-wash s-IgA and serum IgG than those induced by HA-poly(I:C), a reported potent mucosal vaccine, and provided highly efficient protection against lethal doses of virus challenge in mice. Anti-HA-specific serum IgG levels induced by HA-SF-10 were almost equivalent to those induced by subcutaneous immunization of HA twice. Intranasal administration of HA-SF-10 induced balanced anti-HA-specific IgG1 and IgG2a in sera and IFN-γ- and IL-4-producing lymphocytes in nasal cavity without any induction of anti-HA IgE. The results suggest that HA-SF-10 is a promising nasal influenza vaccine and that SF-10 can be supplied in large quantities commercially.

Effects of chain length of an amphipathic polypeptide carrying the repeated amino acid sequence (LETLAKA)(n) on α-helix and fibrous assembly formation.

Polypeptide α3 (21 residues), with three repeats of a seven-amino-acid sequence (LETLAKA)(3), forms an amphipathic α-helix and a long fibrous assembly. Here, we investigated the ability of α3-series polypeptides (with 14-42 residues) of various chain lengths to form α-helices and fibrous assemblies. Polypeptide α2 (14 residues), with two same-sequence repeats, did not form an α-helix, but polypeptide α2L (15 residues; α2 with one additional leucine residue on its carboxyl terminal) did form an α-helix and fibrous assembly. Fibrous assembly formation was associated with polypeptides at least as long as polypeptide α2L and with five leucine residues, indicating that the C-terminal leucine has a critical element for stabilization of α-helix and fibril formation. In contrast, polypeptides α5 (35 residues) and α6 (42 residues) aggregated easily, although they formed α-helices. A 15-35-residue chain was required for fibrous assembly formation. Electron microscopy and X-ray fiber diffraction showed that the thinnest fibrous assemblies of polypeptides were about 20 Å and had periodicities coincident with the length of the α-helix in a longitudinal direction. These results indicated that the α-helix structures were orientated along the fibrous axis and assembled into a bundle. Furthermore, the width and length of fibrous assemblies changed with changes in the pH value, resulting in variations in the charged states of the residues. Our results suggest that the formation of fibrous assemblies of amphipathic α-helices is due to the assembly of bundles via the hydrophobic faces of the helices and extension with hydrophobic noncovalent bonds containing a leucine.

Surfactant protein C is an essential constituent for mucosal adjuvanticity of Surfacten, acting as an antigen delivery vehicle and inducing both local and systemic immunity.

We have reported that Surfacten(®) (St), a bovine pulmonary surfactant free of antigenic c-type lectins, is a useful mucosal adjuvant for nasal vaccination. To prepare ample supplies a synthetic adjuvant that mimics St, we analyzed essential constituents of St for mucosal adjuvanticity. Intranasal inoculation of influenza virus hemagglutinin (HA) vaccine combined with St free of surfactant protein (SP)-C resulted in failure of HA vaccine delivery to dendritic cells and loss of local and systemic immune responses. Naïve bovine SP-C, synthetic human or bovine SP-C peptide reconstituted with three major St lipids restored delivery activity and local and systemic immune responses to levels similar to those of St and provided almost complete protection against lethal doses of influenza virus challenge in mice. The delivery of fluoresceinated HA vaccine to cultured dendritic cells was significantly enhanced by co-administration of St or synthetic adjuvant, and moderately stimulated the expression of MHC class II and CD86. In addition, both St and synthetic adjuvant markedly sustained HA vaccine and achieved a wide antigen distribution in murine nasal cavity. These results suggest that synthetic mucosal adjuvant reconstituted with SP-C peptide and major St lipids is useful for ample supply of the potent mucosal adjuvant as an antigen delivery vehicle for intranasal vaccination.

Influenza vaccine with Surfacten, a modified pulmonary surfactant, induces systemic and mucosal immune responses without side effects in minipigs.

Immune responses and side effects of intranasally administered flu vaccine with the commercial product Surfacten, a modified bovine pulmonary surfactant, were investigated in minipigs. The use of minipigs was based on the anatomical resemblance of nasal lymph nodes, the principal antigen uptake site of respiratory mucosal immunity, between pig and human. Intranasal instillation of HA vaccine adjuvanted with Surfacten elicited significantly higher serum hemagglutination inhibition titers than the antigen alone, with wide cross-neutralizing activities of secretory IgA in nasal washes. No significant induction of inflammatory cytokines or migration of inflammatory cells was observed at the site of immunization or serum after the first immunization. These data suggest the potential usefulness of Surfacten for mucosal vaccination.

[Nasal vaccines].

The nasal route for vaccination offers both mucosal and systemic immunity for the prophylaxis of respiratory diseases. In order to induce maximal protective mucosal immunity, co-administration of mucosal adjuvant is essential. In the present review, we discuss natural pulmonary surfactant and its synthetic compound as safe and effective mucosal adjuvants, particularly for the children. The intranasal administration of influenza hemagglutinin(HA) vaccine with the natural compound or its mimicking synthetic compound induced both anti-HA sIgA and IgG in the airway and in the serum, the efficacy being almost equivalent to cholera toxin. No inflammatory cytokine induction and no inflammatory cell migration were detected in nasal cavity. These data suggest that pulmonary surfactant and its synthetic compound are safe and effective mucosal adjuvants that enhance the protective immunity without incurring a risk of inflammation.

Characterization of the surface activity of a synthetic surfactant with albumin.

We previously reported that a human analogue of pulmonary surfactant protein-C (SP-C), SP-CL16 (6-28), with 23 residues was the most active analogue in a reconstituted lipid mixture and had the shortest chain among the poly leucine analogues examined. In this study, we verified the influence of albumin, a component of serum, on the surface activity of surfactant. Surface activity was measured using the Langmuir-Wilhelmy surface balance (WSB), pulsating bubble surfactometer (PBS), and stable microbubble test (MBT). The surface activity of synthetic lung surfactant (SLS) was only slightly influenced by albumin (0.1-10 mg/ml) as compared with that of a ternary mixture of phospholipids. The ternary mixture of phospholipids showed a decrease in surface activity due to albumin. In particular, SLS did not show interaction of surface activity with albumin in vitro (WSB, PBS, and MBT). In contrast, dipalmitoylphosphatidylcholine/phosphatidylglycerol/palmitic acid had significantly weaker surface activity in the presence of albumin. Surfactant-TA showed interaction of surface activity with albumin in the MBT. The number of stable microbubble increased in the presence of albumin at a concentration of 0.1 mg/ml.

Effects of the human pulmonary surfactant protein-C (SP-C), SP-CL16(6-28) on surface activities of surfactants with various phospholipids.

We previously reported that a human analogue of pulmonary surfactant protein-C (SP-C), SP-CL16(6-28), with 23 residues was the most active analogue in a reconstituted lipid mixture and had the shortest chain among the poly-leucine-analogues examined. There has been little research on the chemical components of synthetic lung surfactants (SLSs). In the present study, we attempted to compare SLS with various phospholipids in surface activity. That is, SP-CL16(6-28) plus various phosphatidylglycerols (PG) were tested for surface activity in a Langmuir-Wilhelmy surface balance (WSB) apparatus and pulsating bubble surfactmeter (PBS). Further, SLSs were examined for biological properties using an animal model of surfactant deficiency, infant respiratory distress syndrome (IRDS), in vivo. Palmitoyl-oleoyl-phosphatidylglycerol (POPG)-SLS exhibited minimum and maximum surface tensions of 1.7 mN/m and 28.6 mN/m in WSB and 8.5 mN/m and 36.2 mN/m in PBS, respectively. Moreover, in the IRDS model, POPG-SLS remarkably improved the lung volume (LV) of a premature lagomorph fetus at LV30 cmH2O and LV5 cmH2O. That is, a significant improvement equal to the LV of a full-term fetus was observed. The level of LV exhibited respiratory improvement equivalent to surfactant-TA. SLS seemed comparable in surface activity with Surfacten (Surfactant-TA), a modified surfactant preparation which has been used for the treatment of RDS.

Characterization of synthetic lung surfactant activity against proinflammatory cytokines in human monocytes.

Our previous study demonstrated that the smallest synthetic peptide with the sequence CPVHLKRLLLLLLLLLLLLLLLL, SP-CL16(6-28), admixed with phospholipid (synthetic lung surfactant, SLS) showed strong surface activity. In this study, we attempted to develop a dual-type surfactant with both anti inflammatory and surface activities. SP-CL16(6-28) was first chemically synthesized and then purified for use by centrifugal partition chromatography. A mixture of SP-CL16(6-28) and phospholipid complex was tested for anti inflammatory activity using the human monocyte cell line THP-1. Whether the suppression of tumor necrosis factor-alpha (TNF-a), interleukin (IL)-8, IL-6, IL-1beta, and macrophage migration inhibitory factor (MIF) was reduced by lipopolysaccharide (LPS) in monocytes was examined. Levels of these cytokines were measured by enzyme-linked immunosorbent assay. It was found that SLS significantly and dose dependently inhibited the secretion of TNF-alpha by THP-1 cells following stimulation with LPS. Dipalmitoylphosphatidylcoline did not inhibit the release of cytokines. These findings suggest that SLS has anti inflammatory activity. Therefore it should be possible to develop a SLS with both anti inflammatory activity and surface activity.