Michael Morr

Cutting Edge: Preferentially the R-Stereoisomer of the Mycoplasmal Lipopeptide Macrophage-Activating Lipopeptide-2 Activates Immune Cells Through a Toll-Like Receptor 2- and MyD88-Dependent Signaling Pathway

Mycoplasmas and their membranes are potent activators of macrophages, the active principle being lipoproteins and lipopeptides. Two stereoisomers of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 (MALP-2) differing in the configuration of the lipid moiety were synthesized and compared in their macrophage-activating potential, the R-MALP being >100 times more active than the S-MALP in stimulating the release of cytokines, chemokines, and NO. To assess the role of the Toll-like receptor (TLR) family in mycoplasmal lipopeptide signaling, the MALP-2-mediated responses were analyzed using macrophages from wild-type, TLR2-, TLR4-, and MyD88-deficient mice. TLR2- and MyD88-deficient cells showed severely impaired cytokine productions in response to R- and S-MALP. The MALP-induced activation of intracellular signaling molecules was fully dependent on both TLR2 and MyD88. There was a strong preference for the R-MALP in the recognition by its functional receptor, TLR2.

The Mycoplasma‐derived lipopeptide MALP‐2 is a potent mucosal adjuvant

The adjuvanticity of MALP‐2, a 2‐kDa synthetic lipopeptide with macrophage‐stimulatory activity, was evaluated in BALB/c mice using β‐galactosidase (β‐gal) as model antigen. When co‐administered with β‐gal by either the intranasal (i.n.) or i.p. route, MALP‐2 (0.5u2004μg) was capable of increasing β‐gal‐specific serum IgG titers by 675–3,560‐fold (i.n.) and 64–128‐fold (i.p.), respectively, as compared to immunization with β‐gal alone. Using MALP‐2, almost maximal IgG responses were already stimulated following the first immunization, and the IgG titers were similar to those observed using 10u2004μg of cholera toxin B subunit (CTB) as adjuvant. The mucosal immune system was also effectively stimulated (p<0.05) when MALP‐2 was administered by the i.n. route (36% and 23% of β‐gal‐specific IgA in lung and vaginal lavages, respectively). The i.n. co‐administration of MALP‐2 stimulated a stronger cellular immune response than CTB, both in submandibular lymph nodes and spleen (p<0.05). The analysis of β‐gal‐specific IgG isotypes and the profiles of cytokines secreted by in vitro re‐stimulated cells showed that co‐administration of MALP‐2 triggered a dominant Th2‐response pattern. A recruitment of B220+ and MAC‐1+ cells with an up‐regulated expression of MHC class I, CD80 (B7.1) and CD54 (ICAM‐1) was observed in nasal associated lymphoid tissues from MALP‐2 treated mice. Taken together, our results demonstrated that the synthetic lipopeptide MALP‐2 represents a very promising adjuvant for the mucosal delivery of vaccine antigens.

Efficient mucosal delivery of the HIV‐1 Tat protein using the synthetic lipopeptide MALP‐2 as adjuvant

A major requirement for HIV/AIDS research is the development of a mucosal vaccine that stimulates humoral and cell‐mediated immune responses at systemic and mucosal levels, thereby blocking virus replication at the entry port. Thus, a vaccine prototype based on biologically active HIV‐1 Tat protein as antigen and the synthetic lipopeptide, macrophage‐activating lipopeptide‐2 (MALP‐2), asa mucosal adjuvant was developed. Intranasal administration to mice stimulated systemic and mucosal anti‐Tat antibody responses, and Tat‐specific T cell responses, that were more efficient than those observed after i.p. immunization with Tat plus incomplete Freunds adjuvant. Major linear B cell epitopes mapped within aa 1–20 and 46–60, whereas T cell epitopes were identified within aa 36–50 and 56–70. These epitopes have also been described in vaccinated primates and in HIV‐1‐infected individuals with better prognosis. Analysis of the anti‐Tat IgG isotypes in serum, and the cytokine profile of spleen cells indicated that a dominant Th1 helper response was stimulated by Tat plus MALP‐2, as opposed to the Th2 response observed with Tat plus incomplete Freunds adjuvant. Tat‐specific IFN‐γ‐producing cells were significantly increased only in response to Tat plus MALP‐2. These data suggest that Malp‐2 may represent an optimal mucosal adjuvant for candidate HIV vaccines based on Tat alone or in combination with other HIV antigens.

A Pegylated Derivative of α-Galactosylceramide Exhibits Improved Biological Properties

The glycolipid α-galactosylceramide (αGalCer) has immunomodulatory properties, which have been exploited to combat cancer, chronic inflammatory diseases, and infections. However, its poor solubility makes αGalCer a suboptimal compound for in vivo applications. In this study, a pegylated derivative of αGalCer is characterized, which exhibits improved physical and biological properties. The new compound, αGalCerMPEG, is water-soluble and retains the specificity for the CD1d receptor of αGalCer. The in vitro stimulatory properties on immune cells (e.g., dendritic cells and splenocytes) are maintained intact, even when tested at a 33-fold lower concentration of the active moiety than αGalCer. NK cells isolated from mice treated with αGalCerMPEG also had stronger cytotoxic activity on YAC-1 cells than those obtained from animals receiving either αGalCer or CpG. Intranasal immunization studies performed in mice showed that αGalCerMPEG exerts stronger adjuvant activities than the parental compound αGalCer when tested at 0.35 vs 11.7 nM/dose. Coadministration of β-galactosidase with αGalCerMPEG resulted not only in high titers of Ag-specific Abs in serum (i.e., 1:512,000), but also in the stimulation of stronger Th2 and secretory IgA responses, both at local and remote mucosal effector sites (i.e., nose, lung, and vagina). The new synthetic derivative αGalCerMPEG represents a promising tool for the development of immune interventions against infectious and noninfectious diseases.