MTADV 5-MER peptide suppresses chronic inflammations as well as autoimmune pathologies and unveils a new potential target-Serum Amyloid A.

Despite the existence of potent anti-inflammatory biological drugs e.g., anti-TNF and anti IL-6 receptor antibodies, for treating chronic inflammatory and autoimmune diseases, these are costly and not specific. Cheaper oral available drugs remain an unmet need. Expression of the acute phase protein Serum Amyloid A (SAA) is dependent on release of pro-inflammatory cytokines IL-1, IL-6 and TNF-α during inflammation. Conversely, SAA induces pro-inflammatory cytokine secretion, including Th17, leading to a pathogenic vicious cycle and chronic inflammation. 5- MER peptide (5-MP) MTADV (methionine-threonine-alanine-aspartic acid-valine), also called Amilo-5MER, was originally derived from a sequence of a pro-inflammatory CD44 variant isolated from synovial fluid of a Rheumatoid Arthritis (RA) patient. This human peptide displays an efficient anti-inflammatory effects to ameliorate pathology and clinical symptoms in mouse models of RA, Inflammatory Bowel Disease (IBD) and Multiple Sclerosis (MS). Bioinformatics and qRT-PCR revealed that 5-MP, administrated to encephalomyelytic mice, up-regulates genes contributing to chronic inflammation resistance. Mass spectrometry of proteins that were pulled down from an RA synovial cell extract with biotinylated 5-MP, showed that it binds SAA. 5-MP disrupted SAA assembly, which is correlated with its pro-inflammatory activity. The peptide MTADV (but not scrambled TMVAD) significantly inhibited the release of pro-inflammatory cytokines IL-6 and IL-1ß from SAA-activated human fibroblasts, THP-1 monocytes and peripheral blood mononuclear cells. 5-MP suppresses the pro-inflammatory IL-6 release from SAA-activated cells, but not from non-activated cells. 5-MP could not display therapeutic activity in rats, which are SAA deficient, but does inhibit inflammations in animal models of IBD and MS, both are SAA-dependent, as shown by others in SAA knockout mice. In conclusion, 5-MP suppresses chronic inflammation in animal models of RA, IBD and MS, which are SAA-dependent, but not in animal models, which are SAA-independent.

Adjuvanted influenza vaccines.

Influenza is one of the most common causes of human morbidity and mortality that is preventable by vaccination. Immunization with available vaccines provides incomplete protection against illness caused by influenza virus, especially in high-risk groups such as the elderly and young children. Thus, more efficacious vaccines are needed for the entire population, and all the more so for high-risk groups. One way to improve immune responses and protection is to formulate the vaccine with antigen carriers and/or adjuvants, which can play an important role in improving immune responses and delivery to antigen-presenting cells, especially for a vaccine like influenza that is based on protein antigens usually administered without a carrier or adjuvant. In this review, the authors present an overview of available vaccines, focusing on research and development of new adjuvants used in influenza vaccines, as well as adjuvanted influenza vaccines aimed to improve immune responses, protection and breadth of coverage for influenza.

A new intranasal influenza vaccine based on a novel polycationic lipid-ceramide carbamoyl-spermine (CCS). II. Studies in mice and ferrets and mechanism of adjuvanticity.

We recently showed that lipid assemblies comprised of a novel polycationic sphingolipid (ceramide carbamoyl-spermine, CCS) are an effective adjuvant/carrier when complexed with cholesterol (CCS/C) for influenza and other vaccines administered parenterally and intranasally (i.n.) in mice. Here we expand these studies to ferrets, an established model of influenza infection. We also address the question of why the CCS/C-based liposomal vaccine (also known as VaxiSome™) in mice is superior to vaccines based on liposomes of other lipid compositions (neutral, anionic or cationic). Ferrets immunized i.n. with CCS/C-influenza vaccine produced significantly higher hemagglutination inhibition (HI) antibody titers compared to ferrets immunized intramuscularly with the unadjuvanted influenza vaccine, indicating that the CCS/C-based vaccine is very immunogenic. Furthermore, the i.n. adjuvanted vaccine was shown to significantly reduce the severity of influenza virus infection in ferrets following homologous viral challenge as determined by weight loss, temperature rise and viral titer. No adverse reactions were observed. Pharmacokinetic and biodistribution studies following i.n. administration in mice of CCS/C-based vaccine showed that both the lipids and antigens are retained in the nose and lung for at least 24h, and it appears that this retention correlates with the superior immunogenicity elicited by the adjuvanted vaccine formulation. The CCS lipid also increases production of cytokines (mainly IFN gamma, IL-2 and IL-12) and co-stimulatory molecules' expression, which might further explain the robust adjuvantation of this liposome-based vaccine.

Femtosecond laser: a new intradermal DNA delivery method for efficient, long-term gene expression and genetic immunization.

A femtosecond laser beam gene transduction (SG-LBGT) system is described as a novel and efficient method of intradermal (i.d.) nonviral gene delivery in mice by permeabilizing cells utilizing femtosecond laser pulses. Using this approach, significant gene expression and efficient dermal transduction lasting for >7 months were obtained. The ability of this new DNA gene transfer method to enhance genetic vaccination was tested in BALB/C mice. A single i.d. injection of a plasmid (10 microg) containing the hepatitis B virus (HBV) surface antigen (HBsAg), followed by pulses of laser, induced high titers of HBsAg-specific antibodies lasting for >210 days and increased levels of IgG1, IgG2a, IFNgamma, and IL-4, indicating the activation of both Th1 and Th2 cells. Moreover, mice vaccinated using the SG-LBGT followed by challenge with pHBV showed increased protection against viral challenge, as detected by decreased levels of HBV DNA, suggesting an efficient Th1 effect against HBV-infected replicating cells. Tumor growth retardation was induced in vaccinated mice challenged with an HBsAg-expressing syngeneic tumor. In most of the parameters tested, administration of plasmid followed by laser application was significantly more effective and prolonged than that of plasmid alone. Tissue damage was not detected and integration of the plasmid into the host genomic DNA probably did not occur. We suggest that the LBGT method is an efficient and safe technology for in vivo gene expression and vaccination and emphasizes its potential therapeutic applications for i.d. nonviral gene delivery.

Mucosal immunization against hepatitis A: antibody responses are enhanced by co-administration of synthetic oligodeoxynucleotides and a novel cationic lipid.

Hepatitis A caused by hepatitis A virus (HAV) transmitted by the fecal-oral route, results in considerable morbidity and economic loss. Mucosal immunization can be more effective than conventional injection at inducing both local and systemic immunity to HAV. Here we show that co-administration of killed HAV with synthetic oligodeoxynucleotides (ODNs) containing CpG sequences, and a novel polycationic sphingolipid (CCS)/cholesterol liposomal delivery system, markedly enhances the HAV-specific antibody response at the intestinal interface, particularly when delivered intrarectally or intranasally, to Balb/c mice at low HAV doses. A mucosally delivered, antigen-sparing HAV vaccine that is easily administered without specialized equipment or personnel, is an attractive alternative for facilitating mass immunization in hepatitis A outbreaks.

A new intranasal influenza vaccine based on a novel polycationic lipid--ceramide carbamoyl-spermine (CCS) I. Immunogenicity and efficacy studies in mice.

Although most pathogens use the mucosal routes for invasion, the majority of currently available vaccines are administered parenterally. Injectable vaccines induce good systemic immunity but often unsatisfactory mucosal immunity. A non-injectable mucosal vaccine, which can be self-administered intranasally, may provide both effective systemic and mucosal immunity and can be used for vaccination of large populations within a short period of time in case of a sudden epidemic. Here, we report on a new intranasal (i.n.) influenza vaccine, based on a novel polycationic sphingolipid, N-palmitoyl D-erythro-sphingosyl carbamoyl-spermine (ceramide carbamoyl-spermine = CCS), having combined carrier and adjuvant activities, which elicits, in mice, strong systemic (serum) and local (lung and nasal) humoral and cellular responses, and provides protective immunity. In a comparative study, we show that both unmodified commercial vaccine and vaccine formulated with neutral or anionic liposomes were poorly immunogenic upon i.n. administration. Of five vaccine formulations based on well-established monocationic lipids in the form of unsized liposomes, three (DC-Chol, DDAB, and DSTAP-based) resulted in low serum and local responses, while two others (DMTAP and DOTAP-based vaccines) induced both systemic and local vigorous Th1+Th2 immune responses. However, only the vaccine formulated with CCS was equivalent or superior to the commercial vaccine co-administered with cholera toxin as an adjuvant. Furthermore, the CCS-based influenza vaccine was highly efficacious following a single or a repeated (x2) i.n. or a single i.m. administration, without an added adjuvant, in both young (2 months) and old (18 months) mice. It elicited high titers of strain cross-reactive hemagglutination inhibition (HI) antibodies, and the high antibody titers and protective immunity persisted for at least 9 months. No systemic adverse effects, and only a mild local inflammatory response, were observed in mice and rabbits vaccinated i.n. with the CCS vaccine formulation. A similar approach may prove efficacious for i.n. vaccination against other pathogens.

The mechanisms controlling the recognition of tumor- and virus-infected cells by NKp46.

The destruction of viral-infected and tumor cells is mediated in part via the lysis receptor of natural killer (NK) cells, NKp46. The nature, however, of its lysis ligands expressed on target cells is poorly defined. Recently, we have identified a novel functional interaction between the lysis receptors NKp46 and NKp44 and the hemagglutinin of influenza and hemagglutinin-neuroaminidase of Sendai viruses. This recognition depends on the sialylation of NKp46 and NKp44 receptors. In this study, we expand the significance of these observations by demonstrating a conserved pattern of NKp46 and NKp44 recognition by various hemagglutinins derived from different viral strains. We further establish that this recognition is direct and mainly mediated via alpha2,6-linked sialic acid carried by NKp46. In addition, we demonstrate that the ability of NKp46 to recognize target cells is confined to the membrane proximal domain, and largely relies on the highly conserved sugar-carrying residue, Thr 225. This residue plays a critical dual role in NKp46 interactions with both viral hemagglutinins and the unknown tumor ligands via different mechanisms. These results may explain the ability of NK cells to kill such a broad spectrum of viral-infected and tumor cells.

Enhanced recognition of human NK receptors after influenza virus infection.

The NK cell cytotoxic activity is regulated by both inhibitory and activating NK receptors. Thus, changes in the expression levels and in the affinity or avidity of those receptors will have a major effect on the killing of target cells. In this study, we demonstrate that the binding of NK-inhibitory receptors is enhanced after influenza virus infection. Surprisingly, however, no change in the level of class I MHC protein expression was observed on the surface of the infected cells. The increased binding was general, because it was observed in both the killer cell Ig-like receptor 2 domain long tail 1 and leukocyte Ig-like receptor-1. The increased binding was functional, was not dependent on the interaction with viral hemagglutinin-neuraminidase, was not dependent on the glycosylation site, and was not abolished after mutating the transmembrane or cytosolic portions of the class I MHC proteins. Confocal microscopy experiments showed increased binding of NK receptor-coated beads to infected cells expressing the appropriate class I MHC proteins. In addition, specific cell-free bead aggregates covered with class I MHC proteins were observed only in infected cells. We therefore suggest that the influenza virus use a novel mechanism for the inhibition of NK cell activity. This mechanism probably involves the generation of class I MHC complexes in infected cells that cause increased recognition of NK receptors.

Immunogenicity and safety of a novel IL-2-supplemented liposomal influenza vaccine (INFLUSOME-VAC) in nursing-home residents.

Influenza and its complications account for substantial morbidity and mortality, especially among the elderly. In young adults, immunization provides 70-90% protection, while among the elderly the vaccine may be only

Immunogenicity and safety of a novel liposomal influenza subunit vaccine (INFLUSOME-VAC) in young adults.

Influenza and its complications account for substantial morbidity and mortality among young adults and especially among the elderly. In young adults, immunization provides 70-90% protection, while among the elderly the vaccine may be only 30-40% effective; hence the need for new, more immunogenic vaccines. We compared the safety and immunogenicity of a novel IL-2-supplemented liposomal influenza vaccine (designated INFLUSOME-VAC) with that of a commercial subunit vaccine and a commercial split virion vaccine in young adults (mean age 28 years) in the winter of 1999-2000. Seventy-three healthy young adults were randomly assigned to be vaccinated intramuscularly with the following: a commercial subunit vaccine (n = 17, group A), INFLUSOME-VAC (n = 36, group B), and a commercial split virion vaccine (n = 20, group C). The three vaccines contained equal amounts of hemagglutinin (approximately 15 microg each) from the strains A/Sydney (H3N2), A/Beijing (H1N1), and B/Yamanashi. INFLUSOME-VAC induced higher geometric mean HI titers and higher-fold increases in HI titers against all three strains, compared with the two commercial vaccines. In addition, seroconversion rates for the A/Sydney and B/Yamanashi strains were significantly higher (P < 0.05) compared with the split virion vaccine, and significantly higher for the three strains compared with the subunit vaccine (69-97% vs 35-65%, P < or = 0.02). Moreover, the anti-neuraminidase response was significantly greater (P = 0.05) in group B vs group A. INFLUSOME-VAC caused mild local pain at the injection site in a significantly higher proportion of the vaccinees (83%). Thus, INFLUSOME-VAC is an immunogenic and safe vaccine in young adults.

Liposomal immunostimulatory DNA sequence (ISS-ODN): an efficient parenteral and mucosal adjuvant for influenza and hepatitis B vaccines.

Synthetic oligodeoxynucleotides (ODNs) containing immunostimulatory sequences (ISS-ODN, also known as CpG-ODNs) have been shown to display in experimental models potent Th1-biassed immunoadjuvant activity upon parenteral or mucosal co-administration with a variety of antigens. In an attempt to potentiate adjuvant activity, and to reduce dose and number of administrations, ISS-ODN was entrapped (up to 90% efficiency) in large (1.5 microm) multilamellar liposomes using a simple and fast (5 min) procedure. Mice were vaccinated once or twice intramuscularly (i.m.) or intranasally (i.n.) with subunit influenza vaccines (consisting of the viral hemagglutinin and neuraminidase, HN) or with hepatitis B surface antigen particles (HBsAg), either non-encapsulated or liposome-encapsulated, together with free or liposomal ISS-ODN (5-25 microg per dose). At 3-12 weeks post-vaccination, the humoral (systemic, mucosal) and cellular responses and protective immunity were assessed. Vaccine formulations containing liposomal ISS-ODN co-administered with either soluble antigen or liposomal antigen (in the same vesicles or in separate vesicles) were up to 30 times more effective than formulations containing un-encapsulated ISS-ODN in inducing: (a) antigen-specific serum and mucosal IgG2a and IgA antibodies; (b) splenocyte proliferative response, cytotoxic activity and IFNgamma production; (c) a DTH response; and (d) protection against virus challenge. The response was Th1-dominant in the influenza model and a mixed Th1+Th2 response in the hepatitis B model. No adverse reactions were noted. Thus, liposomal encapsulation of ISS-ODN further enhances its inherent adjuvant activity.