Bioactive lipids ALIAmides differentially modulate inflammatory responses of distinct subsets of primary human T lymphocytes.

Autacoid local injury antagonist amides (ALIAmides) are a family of endogenous bioactive acyl ethanolamides that include the renowned palmitoyl ethanolamide (PEA), oleoyl ethanolamide (OEA), and stearoyl ethanolamide (SEA), and that are involved in several biologic processes such as nociception, lipid metabolism, and inflammation. The role of ALIAmides in the control of inflammatory processes has recently gained much attention and prompted the use of these molecules or their analogs, and the pharmacologic manipulation of their endogenous levels, as plausible therapeutic strategies in the treatment of several chronic inflammatory conditions. Since chronic inflammation is mainly driven by cells of adaptive immunity, particularly T lymphocytes, we aimed at investigating whether such bioactive lipids could directly modulate T-cell responses. We found that OEA, PEA, and eicosatrienoyl ethanolamide (ETEA) could directly inhibit both T-cell responses by reducing their production of TNF-α and IFN-γ from CD8 T cells and TNF-α, IFN-γ and IL-17 from CD4 T cells. Furthermore, neither SEA nor docosatrienoyl ethanolamide (DTEA) could affect cytokine production from both T cell subsets. Interestingly, unlike OEA and ETEA, PEA was also able to enhance de novo generation of forkhead box P3 (FoxP3)-expressing regulatory T cells from CD4-naive T cells. Our findings show for the first time that specific ALIAmides can directly affect different T-cell subsets, and provide proof of their anti-inflammatory role in chronic inflammation, ultimately suggesting that these bioactive lipids could offer novel tools for the management of T-cell dependent chronic inflammatory diseases.-Chiurchiù, V., Leuti, A., Smoum, R., Mechoulam, R., Maccarrone, M. Bioactive lipids ALIAmides differentially modulate inflammatory responses of distinct subsets of primary human T lymphocytes.

Roles of fatty acid ethanolamides (FAE) in traumatic and ischemic brain injury.

Ethanolamides of long-chain fatty acids are a class of endogenous lipid mediators generally referred to as N-acylethanolamines (NAEs). NAEs include anti-inflammatory and analgesic palmitoylethanolamide, anorexic oleoylethanolamide, stearoylethanolamide, and the endocannabinoid anandamide. Traumatic brain injury (TBI), associated with a high morbidity and mortality and no specific therapeutic treatment, has become a pressing public health and medical problem. TBI is a complex process evoking systemic immune responses as well as direct local responses in the brain tissues. The direct (primary) damage disrupts the blood-brain barrier (BBB), injures the neurons and initiates a cascade of inflammatory reactions including chemokine production and activation of resident immune cells. The effect of TBI is not restricted to the brain; it can cause multi-organ damage and evoke systemic immune response with cytokine and chemokine production. This facilitates the recruitment of immune cells to the site of injury and progression of the inflammatory reaction. Depending on severity, TBI induces immediate neuropathologic effects that, for the mildest form, may be transient; however, with increasing severity, these injuries cause cumulative neural damage and degeneration. Moreover, TBI leads to increased catabolism of phospholipids, resulting in a series of phospholipid breakdown products, some of which have potent biological activity. Ischemia-reperfusion (I/R) injury resulting from stroke leads to metabolic distress, oxidative stress and neuroinflammation, making it likely that multiple therapeutic intervention strategies may be needed for successful treatment. Current therapeutic strategies for stroke need complimentary neuroprotective treatments to provide a better outcome. Prior studies on NAEs have demonstrated neurotrophic/neuroprotective activities across a broad spectrum of cellular and animal models of neurodegenerative and acute cerebrovascular disorders. The present review will summarize our knowledge of the biological role of these lipid signaling molecules in brain and highlights their therapeutic effect from multipotential actions on neuronal cell death and neuroinflammatory pathways.

Palmitoylethanolamide in CNS health and disease.

The existence of acylethanolamides (AEs) in the mammalian brain has been known for decades. Among AEs, palmitoylethanolamide (PEA) is abundant in the central nervous system (CNS) and conspicuously produced by neurons and glial cells. Antihyperalgesic and neuroprotective properties of PEA have been mainly related to the reduction of neuronal firing and to control of inflammation. Growing evidence suggest that PEA may be neuroprotective during CNS neurodegenerative diseases. Advances in the understanding of the physiology and pharmacology of PEA have potentiated its interest as useful biological tool for disease management. Several rapid non-genomic and delayed genomic mechanisms of action have been identified for PEA as peroxisome proliferator-activated receptor (PPAR)-α dependent. First, an early molecular control, through Ca(+2)-activated intermediate- and/or big-conductance K(+) channels opening, drives to rapid neuronal hyperpolarization. This is reinforced by the increase of the inward Cl(-) currents due to the modulation of the gamma aminobutyric acid A receptor and by the desensitization of the transient receptor potential channel type V1. Moreover, the gene transcription-mediated mechanism sustains the long-term anti-inflammatory effects, by reducing pro-inflammatory enzyme expression and increasing neurosteroid synthesis. Overall, the integration of these different modes of action allows PEA to exert an immediate and prolonged efficacious control in neuron signaling either on inflammatory process or neuronal excitability, maintaining cellular homeostasis. In this review, we will discuss the effect of PEA on metabolism, behavior, inflammation and pain perception, related to the control of central functions and the emerging evidence demonstrating its therapeutic efficacy in several neurodegenerative diseases.

Fabrication of calix[4]arene derivative monolayers to control orientation of antibody immobilization.

Three calix[4]arene (Cal-4) derivatives which separately contain ethylester (1), carboxylic acid (2), and crownether (3) at the lower rim with a common reactive thiol at the upper rim were synthesized and constructed to self-assembled monolayers (SAMs) on Au films. After spectroscopic characterization of the monolayers, surface coverage and orientation of antibody immobilized on the Cal-4 derivative SAMs were studied by surface plasmon resonance (SPR) technique. Experimental results revealed that the antibody could be immobilized on the Cal-4 derivatives spontaneously. The orientation of absorbed antibody on the Cal-4 derivative SAMs is related to the SAM's dipole moment. The possible orientations of the antibody immobilized on the Cal-4 derivative 1 SAM are lying-on or side-on, while on the Cal-4 derivative 2 and Cal-4 derivative 3 head-on and end-on respectively. These experimental results demonstrate the surface dipole moment of Cal-4 derivative appears to be an important factor to antibody orientation. Cal-4 derivatives are useful in developing site direct protein chips.

Rutin suppresses palmitic acids-triggered inflammation in macrophages and blocks high fat diet-induced obesity and fatty liver in mice.

PURPOSE: To elucidate the mechanism of rutin in blocking macrophage-mediated inflammation and high fat diet-induced obesity and fatty liver. METHODS: Both in vitro and in vivo approaches were taken in evaluating the effects of rutin on palmitic acids-triggered inflammation in cultured macrophages, and on weight gain and development of fatty liver of mice fed a high fat diet. RESULTS: Palmitic acids increase mRNA levels of pro-inflammatory cytokines, and elevate the production of TNFα in cultured macrophages. Pre-exposure of rutin to cells greatly suppressed these elevations. The suppressed inflammation by rutin was correlated with a decrease in transcription of genes responsible for ER stress and production of reactive oxygen species. In vivo, rutin protects mice from high fat diet-induced obesity, fatty liver and insulin resistance. The protective effects were associated with lack of hypertrophy and crown-like structures in the white adipose tissue, decreased mRNA levels of marker genes for macrophages including F4/80, Cd11c and Cd68, and repressed transcription of genes involved in chronic inflammation such as Mcp1 and Tnfα in white adipose tissue. In addition, rutin increases the expression of genes responsible for energy expenditure in brown adipose tissue including Pgc1α and Dio2. Furthermore, rutin suppresses transcription of Srebp1c and Cd36 in the liver, leading to a blockade of fatty liver development. CONCLUSION: These results suggest that supplementation of rutin is a promising strategy for blocking macrophage-mediated inflammation and inflammation-induced obesity and its associated complications.

Antibacterial activity of sphingoid bases and fatty acids against Gram-positive and Gram-negative bacteria.

There is growing evidence that the role of lipids in innate immunity is more important than previously realized. How lipids interact with bacteria to achieve a level of protection, however, is still poorly understood. To begin to address the mechanisms of antibacterial activity, we determined MICs and minimum bactericidal concentrations (MBCs) of lipids common to the skin and oral cavity--the sphingoid bases D-sphingosine, phytosphingosine, and dihydrosphingosine and the fatty acids sapienic acid and lauric acid--against four Gram-negative bacteria and seven Gram-positive bacteria. Exact Kruskal-Wallis tests of these values showed differences among lipid treatments (P < 0.0001) for each bacterial species except Serratia marcescens and Pseudomonas aeruginosa. D-sphingosine (MBC range, 0.3 to 19.6 µg/ml), dihydrosphingosine (MBC range, 0.6 to 39.1 µg/ml), and phytosphingosine (MBC range, 3.3 to 62.5 µg/ml) were active against all bacteria except S. marcescens and P. aeruginosa (MBC > 500 µg/ml). Sapienic acid (MBC range, 31.3 to 375.0 µg/ml) was active against Streptococcus sanguinis, Streptococcus mitis, and Fusobacterium nucleatum but not active against Escherichia coli, Staphylococcus aureus, S. marcescens, P. aeruginosa, Corynebacterium bovis, Corynebacterium striatum, and Corynebacterium jeikeium (MBC > 500 µg/ml). Lauric acid (MBC range, 6.8 to 375.0 µg/ml) was active against all bacteria except E. coli, S. marcescens, and P. aeruginosa (MBC > 500 µg/ml). Complete killing was achieved as early as 0.5 h for some lipids but took as long as 24 h for others. Hence, sphingoid bases and fatty acids have different antibacterial activities and may have potential for prophylactic or therapeutic intervention in infection.

Protective role of palmitoylethanolamide in contact allergic dermatitis.

BACKGROUND: Palmitoylethanolamide (PEA) is an anti-inflammatory mediator that enhances the activation by anandamide (AEA) of cannabinoid receptors and transient receptor potential vanilloid type-1 (TRPV1) channels, and directly activates peroxisome proliferator-activated receptor-alpha (PPAR-alpha). In mice, 2,4-dinitrofluorobenzene (DNFB)-induced contact allergic dermatitis (CAD) in inflamed ears is partly mediated by the chemokine Monocyte Chemotactic Protein-2 (MCP-2) and accompanied by elevation of AEA levels. No datum is available on PEA regulation and role in CAD. OBJECTIVE: We examined whether PEA is produced during DNFB-induced CAD, and if it has any direct protective action in keratinocytes in vitro. METHODS: Eight- to ten-week-old female C57BL/6J wild-type and CB(1)/CB(2) double knock-out mice were used to measure PEA levels and the expression of TRPV1, PPAR-alpha receptors and enzymes responsible for PEA biosynthesis and degradation. Human keratinocytes (HaCaT) cells were stimulated with polyinosinic polycytidylic acid [poly-(I:C)], and the expression and release of MCP-2 were measured in the presence of PEA and antagonists of its proposed receptors. RESULTS: 2,4-Dinitrofluorobenzene increased ear skin PEA levels and up-regulated TRPV1, PPAR-alpha and a PEA-biosynthesizing enzyme in ear keratinocytes. In HaCaT cells, stimulation with poly-(I:C) elevated the levels of both PEA and AEA, and exogenous PEA (10 microM) inhibited poly-(I:C)-induced expression and release of MCP-2 in a way reversed by antagonism at TRPV1, but not PPAR-alpha. PEA (5-10 mg/kg, intraperitoneal) also inhibited DNFB-induced ear inflammation in mice in vivo, in a way attenuated by TRPV1 antagonism. CONCLUSIONS: We suggest that PEA is an endogenous protective agent against DNFB-induced keratinocyte inflammation and could be considered for therapeutic use against CAD.

Digestion, absorption and tissue distribution of ovalbumin and palmitoyl-ovalbumin: impact on immune responses triggered by orally administered antigens.

Previous work in this laboratory has demonstrated that ovalbumin coupled to palmitoyl residues (palmitoyl-Ova) does not induce oral tolerance. The present study sought to determine whether this coupling affects digestion, absorption and transfer of antigen. Ova and palmitoyl-Ova were shown to be digested differently in vitro by proteolytic enzymes and presented different tissue distribution kinetics after being labelled with (99m)technetium and orally administered to animals. Palmitoyl-Ova remained longer in the stomach, while native Ova was quickly transferred to the gut and other organs. After 3 h, higher levels of palmitoyl-Ova were found in the blood, Peyer's patches, mesenteric lymph nodes, liver and, especially, the spleen, which appears to be essential for immunization with palmitoyl-Ova. In fact, splenectomized mice treated orally with palmitoyl-Ova became tolerant, while tolerance to Ova was not affected. Thus, palmitoyl coupling was demonstrated to affect antigen digestion, absorption and transport. This is the first time that the spleen has been shown to be required for oral immunization with palmitoyl-Ova.

Thiopalmitoylation of myelin proteolipid protein epitopes enhances immunogenicity and encephalitogenicity.

Proteolipid protein (PLP) is the most abundant protein of CNS myelin, and is posttranslationally acylated by covalent attachment of long chain fatty acids to cysteine residues via a thioester linkage. Two of the acylation sites are within epitopes of PLP that are encephalitogenic in SJL/J mice (PLP(104-117) and PLP(139-151)) and against which increased immune responses have been detected in some multiple sclerosis patients. It is known that attachment of certain types of lipid side chains to peptides can result in their enhanced immunogenicity. The aim of this study was to determine whether thioacylated PLP peptides, as occur in the native protein, are more immunogenic than their nonacylated counterparts, and whether thioacylation influences the development of autoreactivity and experimental autoimmune encephalomyelitis. The results show that in comparison with nonacylated peptides, thioacylated PLP lipopeptides can induce greater T cell and Ab responses to both the acylated and nonacylated peptides. They also enhanced the development and chronicity of experimental autoimmune encephalomyelitis. Synthetic peptides in which the fatty acid was attached via an amide linkage at the N terminus were not encephalitogenic, and they induced greater proportions of CD8+ cells in initial in vitro stimulation. Therefore, the lability and the site of the linkage between the peptide and fatty acid may be important for induction of encephalitogenic CD4+ T cells. These results suggest that immune responses induced by endogenous thioacylated lipopeptides may contribute to the immunopathogenesis of chronic experimental demyelinating diseases and multiple sclerosis.

Preparation of immune stimulating complexes (ISCOMs) as adjuvants.

Purified proteins are often poorly immunogenic and in such cases the induction of primary immune responses requires use of an adjuvant. The immune stimulating complex (ISCOM) has a unique ability to provoke a full range of immune response to protein antigens, after both parenteral and oral immunization. This unit describes techniques for incorporating proteins into the ISCOM structure, a process that requires the presence of exposed hydrophobic regions on the protein. The basic protocol outlines a method for preparation of ISCOMs containing inherently nonhydrophobic proteins, to which palmitic acid has been attached covalently. Two alternate protocols are given that do not require covalent modification of the protein. In the first, hydrophobic groups are revealed by acid treatment of the protein. The second describes preparation of ISCOMs containing integral membrane proteins that therefore possess a hydrophobic transmembrane domain.

Purification and characterization of Streptococcus pneumoniae palmitoylated pneumococcal surface adhesin A expressed in Escherichia coli.

All Streptococcus pneumoniae isolates tested to date express a species-common lipoprotein designated as pneumococcal surface adhesin A (PsaA). This protein is cell-associated, hydrophobic, immunogenic, and genetically conserved. It is currently under investigation as a potential component in third-generation pneumococcal vaccine formulations. To overcome the problem of low-level expression of native hydrophobic PsaA in S. pneumoniae, and also of the recombinant PsaA (rPsaA) in Escherichia coli, we generated a stable E. coli construct expressing functional palmitoylated rPsaA ( approximately 10 mg/l of fermentation culture) using Borrelia burgdorferi outer surface protein A (OspA, a hydrophobic lipoprotein) signal peptide. By Western blot analysis, the chimeric rPsaA ( approximately 34 kDa) was detected in the cell lysate using anti-PsaA antibodies. It was partially purified by extracting the cell pellet with PBS/Triton X(R)-114 buffers, followed by anion exchange filter chromatography. A trypsin digestion profile of rPsaA closely resembled that of the native protein, as revealed by SDS-PAGE/silver staining. Lipidation of rPsaA was confirmed by labeling recombinant E. coli cells with [(3)H] palmitic acid and analyzing the labeled E. coli cells by Western blotting coupled with autoradiography. Further, analysis of purified rPsaA by mass spectrometry (MALDI-TOF) revealed a heterogenous spectrum with a major peak (M+H)(+1) of mass 33,384 Da (theoretical mass of palmitoylated rPsaA=33,361 Da). Purified rPsaA was immunogenic in CBA/NCAHN-XID female mice following intranasal immunization with or without adjuvant, as determined by measurement of anti-PsaA serum IgG levels. These anti-PsaA antibodies reacted with both native and rPsaA polypeptides. Our data strongly suggest that E. coli-expressed rPsaA is palmitoylated and closely resembles the native protein in structure and immunogenicity. It was also observed to elicit measurable protection against nasopharyngeal carriage with S. pneumoniae.

Priming for virus-specific CD8+ but not CD4+ cytotoxic T lymphocytes with synthetic lipopeptide is influenced by acylation units and liposome encapsulation.

Synthetic peptides of the herpes simplex virus glycoprotein B synthesized either as a free form or derivatized with one (PAM1) or three palmitic acids (PAM3Cys) were used to assess the in vivo priming efficacy of high affinity virus-specific CTL induction. The peptide and its derivatives were delivered in vivo with or without liposome encapsulation. Neither the free peptide nor the PAM1 derivative primed for high affinity virus specific CD8+ CTL induction, whether delivered via liposomes or not. On the other hand, the PAM3Cys derivative was able to prime for low levels of high affinity virus specific CD8+ CTL induction in the absence of liposome encapsulation. However, the efficiency of virus-specific CD8+ CTL induction with PAM3Cys derivative was enhanced following encapsulation in the liposomes. In contrast, all forms of the peptides induced both CD4+ T cell proliferative response as well as high affinity virus-specific CD4+ CTL. In addition, the efficiency of the PAM3Cys derivative to prime for CD4+ or CD8+ CTL was found to be influenced by the liposome encapsulation. When delivered via liposomes, the PAM3Cys derivative effectively primed for CD8+ CTL. However, liposomal delivery was not necessary for efficient priming for CD4+ CTL induction. Thus, both the acylation units as well as liposomal delivery appear to influence the in vivo priming of CD4+ and CD8+ T cell responses with synthetic peptides.

Immune response against the murine mdri protein induced by vaccination with synthetic lipopeptides in liposomes.

Intrinsically, or after exposure to chemotherapeutic drugs, many cancer cells overexpress a class of high molecular weight membrane glycoproteins associated with the multidrug resistance (mdr) of these cells. This report describes an immunization protocol eliciting autoantibodies specific to extracellular epitopes of the murine mdr 1 P-glycoprotein (Pgp). Synthetic peptides with the sequences of extracellular loops of murine Pgp were covalently coupled with four palmitic acid moieties per peptide molecule. These "lipopeptides" were reconstituted in the bilayer of liposomes containing lipid A and used to immunize mice. Antibodies against the lipopeptides corresponding to loop 2 and 4 were elicited in sera of immunized mice. They reacted specifically with extracellular epitopes of the naturally occurring murine Pgp. After interaction with resistant cancer cells, the antibodies induced an average 50% increase in cellular accumulation of doxorubicin and Bodipy-verapamil. In the presence of these antibodies the resistance of L1210 mdr cells was reduced from an LD50 of 4 x 10(-5) M to 5 x 10(-7) M doxorubicin.

Synthesis of alpha,alpha-trehalose 2,3- and 2,3'-diesters with palmitic and stearic acid: potential immunoreactants for the serodiagnosis of tuberculosis.

Regioselective monoacylation, by the stannylation method, of 4,6:4',6'-di-O-benzylidene-alpha,alpha-trehalose with palmitoyl or stearoyl chloride afforded the 2-palmitate and 2-stearate of the diacetal, whereas partial diacylation led to the corresponding 2,3'-dipalmitate and 2,3'-distearate. Protection of the monoesters in the 2',3' positions by cyclizing silylation with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, followed by acylation of the silyl ethers, gave the fully protected 2,3-dipalmitate, 2,3-distearate, and 2-palmitate-3-stearate. Small proportions of other isomers and triesters were also produced in these reactions. Desilylation and debenzylidenation of the diesters finally furnished 2,3- and 2,3'-di-O-palmitoyl-2,3- and 2,3'-di-O-stearoyl-, and 2-O-palmitoyl-3-O-stearoyl-alpha,alpha-trehalose.

Palmitic acid conjugation of a protein antigen enhances major histocompatibility complex class II-restricted presentation to T cells.

The effect on antigenicity of covalent attachment of lipid groups to a protein antigen was investigated. Coupling of palmitic acid to ovalbumin (OVA) enhanced major histocompatibility complex (MHC) class II-restricted presentation to most OVA-specific murine T-cell clones in vitro. The enhanced antigenicity of palmitoylated antigen was localized to the level of presentation of the synthetic peptide epitope, OVA 323-339. T-cell responses to palmitoylated antigen were more difficult to block with anti-MHC class II antibodies than responses to native antigen. However, T-cell proliferation to palmitoyl (p)-OVA and native (n)-OVA were blocked equally by anti-CD4 antibodies. Taken together, the results suggest that lipid conjugation of a protein antigen leads to the formation of a lipopeptide T-cell epitope with increased affinity of binding to MHC class II and/or T-cell receptor (TcR). These results have implications for the design of synthetic peptide vaccines.

Identification of T-cell epitopes without B-cell activity in the first and second conserved regions of the HIV Env protein.

We have previously hypothesized that an effective vaccine against HIV should elicit cell-mediated immunity without antiviral antibody production. As a first step towards this goal we have identified potential T-cell epitopes, without B-cell activity against the native protein, from the first and second conserved sequences, and from three functionally important regions of the HIV-1 envelope protein gp160. For this approach, short peptide sequences selected by established computer programs were synthesized and chemically modified to generate either polymers with disulfide bonds, or micelles with two palmitic acid residues attached to the amino-terminal lysine. In both configurations several peptides were immunogenic without the need for coupling to carrier molecules. Of the 19 peptides we tested in our present studies, seven induced good T-cell proliferative response in mice representing four major histocompatibility complex haplotypes. None of these seven peptides produced antibodies that could recognize the envelope protein gp160.

Lectins and antibodies as tools for studying cellular interactions.

Specific interactions between multiple cell types are critical for a variety of processes central to the development, homeostasis and immune defense of multicellular organisms. Studies designed to elucidate how cells communicate through physical encounters have exploited exogenously supplied factors to bypass intrinsic recognition mechanisms and facilitate cellular conjugation. In this review, we compare the relatively nonspecific agglutinating properties of lectins and the selective cell targeting capabilities of antibodies and bispecific antibody constructs for studying cell-cell interactions in immunobiology. In addition, we discuss a novel system for inducing cellular interactions which closely resembles native receptor-mediated conjugation. In this system, surrogate receptors promote specific cell-cell interactions without hindering endogenous receptor-ligand interactions at the cell-cell interface which may be important in mediating physiologic cellular responses.

Palmitate-derivatized antibodies can function as surrogate receptors for mediating specific cell-cell interactions.

The observation that exogenously supplied agents can bypass intrinsic recognition mechanisms and facilitate cellular conjugation has led to valuable insights into the mechanisms of cell function. A common feature of currently available cellular conjugation agents is their reliance on endogenous membrane molecules on both cell types as anchors for cellular interactions. In this report, we describe a method for incorporating palmitate-derivatized antibody molecules onto cell membranes where they function as 'surrogate receptors' (SR) for mediating specific cellular interactions. In this system, SR are attached to the plasma membrane by insertion of the palmitate hydrocarbon chains into the outer leaflet of the phospholipid bilayer. Therefore, the palmitate anchor bypasses the requirement for FcR or other endogenous membrane proteins in antibody-dependent cellular conjugation. Due to this mode of attachment, which is similar to that of phosphatidylinositol (PI) anchored proteins, SR-mediated cellular interactions are likely to be reminiscent of native receptor-induced conjugation, enabling SR to cooperate with endogenous target recognition structures in receptor-ligand interactions at the cell-cell interface.

A synthetic peptide induces long-term protection from lethal infection with herpes simplex virus 2.

Immunization against viral pathogens is generally directed toward the induction of virus neutralizing antibody (VNA) and the maintenance of the potential for a second-set (IgG) response. Indeed, an elevated level of specific antibody is considered a reliable clinical indicator that a state of immunity exists in the host. However, in the case of herpes simplex virus (HSV), the presence of circulating VNA does not necessarily correlate with protection. Thus, it has been found that secondary infections occur in individuals even with high neutralizing titers to HSV, suggesting that antibody to the virus may be useless or even deleterious. In consideration of these facts, we were interested in inducing a T cell response to HSV. We had already shown that synthetic peptides corresponding to the NH3-terminal region of the glycoprotein D (gD) molecule of HSV could induce a strong T cell response when injected into mice, but did not, by themselves, confer protection. In this report, we examined the ability of peptides, covalently coupled to palmitic acid and incorporated into liposomes, to induce virus-specific T cell responses that confer protection against a lethal challenge of HSV-2. We have demonstrated that long-term protective immunity is achieved with a single immunization in the absence of neutralizing antibody when antigen is presented in this form. Furthermore, T cells but not serum from such immune mice can adoptively transfer this protection.

Immunogenicity of a synthetic HBsAg peptide: enhancement by conjugation to a fatty acid carrier.

Effective immunization with short polypeptide antigens has typically only been possible when the peptide is conjugated to a large carrier substance, usually a protein. Such immunizations suffer from difficulties in producing conjugates of reliable composition, and from unwanted anti-carrier immune responses. When a chemically synthesized peptide, bearing hepatitis B virus a-determinant specificity, was conjugated to a dipalmityl-lysine moiety, a significant improvement in anti-hepatitis B surface antigen response was obtained, in comparison to the corresponding peptide-keyhole limpet hemocyanin conjugate. Dipalmityl lysyl peptide conjugates are readily made by standard Merrifield synthesis procedures, and are relatively free of byproducts that might cause unwanted immune responses. Gel filtration experiments suggest that the conjugates form large aggregates, possibly micelles, which may play a significant role in the enhancement of the anti-peptide response. These properties suggest that fatty acid conjugation may be a useful procedure for producing chemically synthesized peptide vaccines.