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In previous studies we have shown that lipopeptides constitute potent immunoadjuvants in mice, rabbits and other species: in parenteral immunization, lipopeptide adjuvants were comparable, or in some cases superior to Freund's adjuvant, and were devoid of the side effects of this additive. Here we demonstrate that lipopeptides also constitute adjuvants for mucosal immunizations. The serum antibody responses against the wheat storage protein gliadin, the bee venom constituent melittin, or the hen egg protein ovalbumin could in most cases be enhanced more than 100-fold by the lipopeptide P3CSK4, applied via the nasal route. An enhanced specific antibody level could also be detected in supernatants of cell cultures prepared from spleens, Peyer's patches, lungs and mesenteric lymph nodes of immunized mice. Moreover, the lipopeptide P3CSK4 enhanced chemiluminescence in mouse spleen cells and peritoneal macrophages in vitro, indicating a macrophage-activating effect. Finally, nasal application of lipopeptide increased protection against a lethal infection of influenza. Our findings are of importance for the improvement of immunizations and might lead to more effective vaccines.

To evaluate the ability of the lipotripeptide P3CSS to increase peptide-specific immune responses in vivo, we immunized mice from different inbred strains (BALB/c, C3H/HeJ, C57BL/6) with the 22-mer lipopeptide conjugates P3CSS-[RT-(522-543)] and P3CSS-[RT-(528-549)] of HIV-1 reverse transcriptase (RT) which included an immunodominant Th epitope [i.e. RT-(528-543)] characterized previously. Analysis of T and B cell responses to these lipopeptide conjugates indicated that specific Th responses could be readily induced in vivo. The peptide segments could also efficiently prime mice for secondary recognition of native RT. The use of shorter peptides permitted a delineation of the minimal T cell recognition site of this RT C-terminal region [i.e. RT-(528-540)]. Close to this T cell epitope we identified a B cell determinant containing the motif EQVD [RT-(546-549)] which was recognized in three different strains of mice (H-2b, H-2d and H-2k). A comparison with X-ray analysis of the C-terminal region of HIV-1 reverse transcriptase indicated exposed positions of these Th and B cell epitopes. Both the presence of T and B cell sites and its limited polymorphism make the region RT-(528-549) a promising candidate for vaccine design. The use of the P3CSS adjuvant/carrier principle as a nontoxic adjuvant may be of major importance in the development of vaccines applicable to humans.

Two lipopeptide analogues of the Escherichia coli lipoprotein rendered water-soluble by polyoxyethylene were tested for mitogenicity in vitro in murine and human B lymphocytes and for adjuvant activity in vivo in mice. These highly amphiphilic lipopeptides retained the biological activity other lipopeptides usually exerted which supports the hypothesis of specific interactions of lipopeptides with membranes of reactive cells. The activation of human B lymphocytes by these lipopeptides was much less pronounced compared to that of murine cells. However, given in combination with anti-CD40 antibodies plus interleukin-4, human B lymphocytes could synergistically be stimulated to proliferate. As an adjuvant, the polyoxyethylene linked lipopeptides were almost as potent as Freund's adjuvants and other basic lipopeptides. Being water-soluble, these novel analogues are easy to apply and they are suitable for field studies as adjuvants when sonication can not usually be provided.

Mycelia from several strains of Streptomyces were potent B-lymphocyte mitogens for spleen cells from 3 inbred mouse strains (BALB/c, C3H/HeJ, C3H/nu/nu), inducing lymphocyte proliferation and differentiation into immunoglobulin-secreting cells. Streptomyces mycelia were also mitogenic towards human peripheral blood lymphocytes, where maximal stimulation was found on days 5 to 7. On the other hand, human lymphocytes incubated with the mycelia for 3 days, but not the supernatants of these cultures, strongly inhibited mixed leukocyte reactions. Irradiation experiments suggest the induction of both radioresistant and radiolabile suppressor-cell populations by the components.

The lipopeptide Pam3Cys-Ser, a synthetic analogue of the N-terminal part of bacterial lipoprotein, constitutes a potent activator for B lymphocytes, monocytes/macrophages and several lymphoid cell lines. We applied the novel method of electron energy loss spectroscopy (EELS) to determine, after stimulation, the distribution of the activator within the cell compartments of the lipopeptide sensitive cell line BCL1. Our results show that the lipopeptide, 20 min after the addition to the cell culture, was found at different locations within the cell: A major amount of the mitogen was found in the plasma membrane. Remarkably, considerable amounts of the activator were also found on the cytoplasm, the nuclear membrane, and the nucleus. After 24 h, a substantial amount of the lipopeptide was still present within the cells. These findings should help to elucidate the molecular mechanism of lymphocyte stimulation by lipopeptides. The novel method of EELS, which was demonstrated here using lipopeptides as examples, constitutes a valuable tool of localizing any given compounds such as growth factors or drugs within cells.

The synthetic lipopeptide Pam3Cys-Ser-Ser-Asn-Ala, an analogue of the N-terminal part of bacterial lipoprotein, constitutes a potent macrophage and B lymphocyte activator. In the macrophage cell-line P388D1 Pam3Cys-Ser-Ser-Asn-Ala stimulated phosphoinositol turnover, whereas in small resting lymphocytes no enhanced turnover was observed. Upon lipopeptide stimulation, a translocation of PKC from the cytosol to the plasma membrane was found in the cell line P388D1 but not in lymphocytes. Substituting lipopeptide for diacylglycerol shows that Pam3Cys-Ser-Ser-Asn-Ala leads to an activation of protein kinase C at Ca2+ concentrations of 0.5 mM. Thus, mitogenic lipopeptides constitute novel tools for investigating the molecular mechanism of transmembrane signaling in leukocyte activation.

S-(2,3-Bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-cysteinyl-( S)-serine (Tripam-Cys-Ser), a synthetic analogue of the N-terminal part of bacterial lipoprotein, constitutes a potent B-lymphocyte activator, inducing mitosis and immunoglobulin production, and is an adjuvant of the humoral immune response. The product was covalently coupled to a nonimmunogenic oligopeptide, representing an extracytoplasmic sequence of the receptor for the epidermal growth factor (EGF-R amino acids 516-529: Asn-Leu-Leu-Glu-Gly-Glu-Pro-Arg-Glu-Phe-Val-Glu-Asn-Ser). The resulting conjugate was able to induce a specific antibody response 2 wk after a single i.p. administration in mice, as measured by ELISA assays. Controls performed by injecting either adjuvant or oligopeptide alone, or a mixture of both compounds, elicited only a marginal stimulation of the specific antibody response. The conjugate was also immunogenic in vitro. Synthetically derived lipopeptides, thus, constitute powerful agents for turning substances of low immunogenicity into potent immunogens, after conjugating them covalently to the adjuvants. This technique should have a wide application in the preparation of conventional and monoclonal antibodies, as well as for the design of synthetic vaccines.

Porins were prepared from the outer membrane of Salmonella typhimurium and consisted of three polypeptides with similar molecular ratios around 40 kDa, and isoelectric points around 4.7. They constituted potent polyclonal activators for murine B lymphocytes. The compounds were mitogenic towards splenic lymphocytes of lipopolysaccharide responder Balb/c, non-responder (C3H/HeJ), and athymic (Balb/c nu/nu) mouse inbred strains, as measured by 3H-thymidine incorporation into DNA. Furthermore, porin-activated B cells differentiated into immunoglobulin-secreting cells, as was measured by an ELISA test. The porins will be valuable in elucidating the molecular mechanism of lymphocyte activation in bacterial infections.

Lipoprotein from the outer membrane of Escherichia coli and its synthetically prepared N-terminal lipopeptide segments Pam3Cys-Ser-Ser-Asn-Ala and Pam3Cys-Ser, as well as lipoprotein from other Enterobacteriaceae, constitute potent polyclonal B lymphocyte activators. Here, we demonstrate that these compounds were also able to stimulate human and murine leukocytes: in murine macrophages, we could show the induction of interleukin 1 release by the mitogens, as measured in the thymocyte proliferation assay. Moreover, murine peritoneal exudate cells were stimulated to secrete prostaglandins E2 (PGE2) and F2 alpha (PGF2 alpha). The effect of Pam3Cys-Ser on the murine macrophage cell line P388D1 was also tested: the compound induced an increase in proliferation, as measured by a thymidine incorporation assay. In addition, the cell line could be induced to release IL 1 into the supernatant. Correspondingly, induction of IL 1 release could also be demonstrated in human mononuclear cells. Our results demonstrate that the two novel synthetic lipopeptides are potent stimulators for human monocytes and murine macrophages. These findings may be important for the elucidation of the role of these bacterial surface components in the course of bacterial infections.

Two analogues of the N-terminal part of bacterial lipoprotein, S-[2,3-bis(palmitoyloxy)-(2RS)propyl]-N-palmitoyl-(R)-cysteine methyl ester ("tripalmitoyl cysteine") and S [-2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteinyl-(S)-seryl-(S)-seryl-(S)-asparaginyl-(S)-alanine ("tripalmitoyl pentapeptide") were synthesized and tested for mitogenic activity. The compounds were potent mitogens towards mouse spleen cell cultures, as measured by 3H-thymidine incorporation and by hemolytic plaque assays. This activity was not dependent on the presence of serum. Tripalmitoyl pentapeptide had little, if any, effect on thymocytes. When injected intravenously into Balb/c mice, the synthetic compounds induced splenomegaly and polyclonal B-cell activation, the latter was evident from an increase in the number of plaque-forming cells against trinitrophenylated sheep red blood cells. Thus, a synthetic fragment of a bacterial surface component was shown to exhibit marked biological activity in vitro as well as in vivo.

The effects of ultrasonication, electrodialysis, and centrifugation through sucrose gradients on the mitogenicity of the lipoprotein (LP) from the outer membrane of Escherichia coli were studied in several inbred mouse strains. LP is not readily soluble in aqueous solution. By ultrasonication for 1 minute we obtained LP aggregates which were fully mitogenic; prolonged sonification had no influence on activity. Solubilization in 4% sodium dodecyl sulphate (SDS) and centrifugation into sucrose gradients yielded different aggregation forms of LP, which were B-lymphocyte mitogens towards LPS non responder C3H/HeJ mouse splenocytes, and towards congenitally athymic nude mice. By electrodialysis, LP could be transformed into different salt forms. The TEA-salt of LP exhibited enhanced mitogenicity. The results suggest that the mitogenicity of lipoprotein is widely independent from the aggregational state in which it is offered to the cells.