Inhibition of human hepatitis B virus (HBV) by a novel non-nucleosidic compound in a transgenic mouse model.

BAY 41-4109 is a member of a class of heteroaryl-pyrimidines that was recently identified as potent inhibitors of human hepatitis B virus (HBV) replication. We have investigated the antiviral activity of BAY 41-4109 (methyl (R)-4-(2-chloro-4-fluorophenyl)-2-(3,5-difluoro-2-pyridinyl)-6-methyl-1,4-dihydro-pyrimidine-5-carboxylate) in HBV-transgenic mice (Tg [HBV1.3 fsX(-)3'5']). Bay 41-4109 was administered per os using different schedules (b.i.d. or t.i.d. for up to 28 days) and dosages ranging from 3 to 30 mg/kg. The compound reduced viral DNA in the liver and in the plasma dose-dependently with efficacy comparable to 3TC. In contrast to 3TC-treated mice, we found a reduction of cytoplasmic hepatitis B virus core antigen (HBcAg) in liver sections of BAY 41-4109-treated mice, which indicated a different mode of action. Pharmacokinetic studies in mice have shown rapid absorption, a bioavailability of 30% and dose-proportional plasma concentrations. We conclude that BAY 41-4109 is a new anti-HBV drug candidate.

Development of resistance and perspectives for future therapies against hepatitis B infections: lessons to be learned from HIV.

Several first-generation nucleoside analogues have been tested against chronic hepatitis B virus (HBV) infection, but trials were unsuccessful or accompanied by toxicity. Recently, oral second-generation nucleoside analogues have been developed that have potent activity against HBV. The best-studied compound so far is lamivudine ((-)2'-deoxy-3'-thiacytidine; 3TC). Lamivudine is an inhibitor of reverse transcriptase (RT) activity and is in clinical use in human immunodeficiency virus (HIV)-infected individuals. As several studies on the use of lamivudine for hepatitis B show, the development of resistance in the viral polymerase under lamivudine treatment, however, causes a significant clinical problem. All other drugs in advanced clinical development for HBV are nucleosides; cross-resistance is therefore expected in most cases. The history of HIV treatment demonstrates that new classes of drugs, the protease inhibitors and non-nucleosidic inhibitors of RT, allowed for a longer-term clinical benefit when used in combination with nucleoside analogues. The development of non-nucleosidic compounds with different modes of action therefore appears very important for the treatment of chronic hepatitis B as well.

Ceramide-independent CD28 and TCR signaling but reduced IL-2 secretion in T cells of acid sphingomyelinase-deficient mice.

Ceramide generated by lysosomal acid sphingomyelinase (aSMase) has been proposed to contribute to CD28 co-stimulatory signaling pathways. We used an aSMase-deficient mouse line (asmase-/-) to elucidate the role of the aSMase in splenocytes stimulated with either a combination of anti-CD3 and anti-CD28 antibodies, the lectin concanavalin A (Con A) or the superantigen staphylococcal enterotoxin B. All stimuli were shown to induce IL-2 expression, Con A additionally triggered the expression of high-affinity IL-2 receptor. However, in asmase-/- mice secretion of IL-2 was significantly reduced, whereas the intracellular IL-2 levels were elevated. Proliferation of anti-CD3/anti-CD28 or Con A-stimulated aSMase-deficient splenocytes was reduced up to 50% after 72 h in comparison to wild-type cells. We conclude that ceramide generated by aSMase is not involved in CD28 signal transduction, but rather a perturbation of the secretory system is responsible for the impaired proliferation of aSMase-deficient splenocytes.

Biological activity of the Escherichia coli lipoprotein: detection of novel lymphocyte activating peptide segments of the molecule and their conformational characterization.

The lipoprotein (LP) from Escherichia coli and other Enterobacteriaceae as well as lipoprotein derived lipopeptides constitute potent B lymphocyte mitogens. Several synthetic peptide segments of the lipoprotein were tested for mitogenic activity towards splenocytes of BALB/c mice. Mitogenic effects were observed for the lipoprotein fragments LP-(2-15), LP-(2-20), LP-(2-25), and LP-(33-47), and a less pronounced effect was determined for LP-(2-10). The mitogenicity of the peptides could be further enhanced by their attachment to the lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)- cysteine (Pam3Cys). The additional insertion of 6-aminohexanoic acid (Ahx) between the peptide segments and Pam3Cys further increased mitogenicity. Thus, in addition to the known mitogenic N-terminal lipopeptide of lipoprotein, additional peptide segments of the molecule were shown to exhibit a biological activity.

MHC/peptide binding studies indicate hierarchy of anchor residues.

MHC class I molecules present octa- or nonapeptides derived from cellular proteins. Such peptides adhere to strict rules, which are individual to each MHC allele. Synthetic peptides conforming to these rules or peptides being at variance at critical residues were assayed for binding to MHC class I molecules. The binding assay employed the peptide-induced stabilization of MHC molecules of RMA-S cells. The data indicate that most proline-free peptides conforming to the allele-specific motifs of Kb or Db bind to the respective molecules, whereas peptides missing only one of the two allele-specific anchor residues lost their capacity to stabilize class I molecules on RMA-S cells. The residues allowed at anchor positions of the Kb motif are not equal in their binding efficiency and can be ordered in a hierarchic row. Residues at nonanchor positions may also influence efficiency of peptide binding or may require deviations from the standard peptide length.

Identification and quantification of a naturally presented peptide as recognized by cytotoxic T lymphocytes specific for an immunogenic tumor variant.

The target antigen recognized by H-2Kd-restricted cytotoxic T lymphocytes (CTLs) specific for a mutagen-induced antigen on DBA/2-derived tumor P815 was identified as the product of a normal cellular gene encompassing a point mutation. Using synthetic peptides, the epitope recognized by these CTLs was narrowed down to be contained within the undecamer KYQAVTTTLEE, incorporating the point mutation. The allele-specific peptide motif for H-2Kd molecules allowed us to predict the peptide naturally presented by the tumor cells to be the nonamer KYQAVTTTL. Isolation of the natural tum(-)-specific peptide from P198.3 tumor cells and biochemical comparison with the synthetic nonamer confirmed the prediction. This natural nonapeptide is represented by approximately 100 copies per tumor cell.

Synthetic peptide libraries in the determination of T cell epitopes and peptide binding specificity of class I molecules.

Major histocompatibility complex (MHC) class I molecules combine with short peptides of defined length and sequence. Here we describe an approach that may be used in the analysis of peptide preference of different allelic MHC class I molecules, and in the determination of T cell epitopes. We produced synthetic "peptide libraries" of limited complexity by standard peptide chemistry. Using these peptide mixtures we show that H-2 Kb molecules can accommodate both 8- and 9-residue peptides, whereas Db molecules are unable to combine with peptides shorter than 9 amino acids present in these libraries. When these peptide mixtures are used to provide "fingerprints" of Db molecules and mutants thereof, both loss and gain of the ability to combine with certain peptides is observed. For the Kbm1 mutant a strong influence of amino acid substitutions in class I molecules on the peptides selected is observed. In these synthetic peptide mixtures, the presence of a specific T cell epitope, known to be represented once, can be detected. This approach may be extended to the identification of new T cell epitopes from larger peptide libraries.

Preferred size of peptides that bind to H-2 Kb is sequence dependent.

The identification of naturally processed viral peptides reveals that major histocompatibility complex (MHC) class I epitopes are composed of nine or eight amino acid residues. Peptides eluted from H-2 Kb MHC class I molecules have been suggested, as a class, to be eight amino acid residues long. To assay for peptide-class I interactions, a stabilization assay described for surface labeled "empty" class I molecules was employed, but on biosynthetically labeled class I molecules. The Sendai virus nucleoprotein-derived octapeptide APGNYPAL does not bind and stabilize Kb molecules, whereas other octameric Kb-restricted peptides and the nonameric peptide FAPGNYPAL interact stably. We attribute the failure of Sendai octamer binding to the presence of proline in position two: replacement of proline renders the resulting octamers as efficient as FAPGNYPAL for binding and stabilization of H-2 Kb. Substitution of glycine in position three of APGNYPAL slightly improves its Kb stabilizing capacity. Iodination of the tyrosine residue significantly alters the binding properties of the nonamer peptide. We conclude that the length of epitopes as selected by the class I Kb molecule is influenced by their sequence and suggest that proper positioning of the NH2 terminus of peptides is essential for class I stabilizing properties. The ability to stabilize newly synthesized "empty" class I molecules with peptide argues against an involvement of beta 2 microglobulin exchange in the experiments described here.

Fine specificity of cytotoxic T lymphocytes primed in vivo either with virus or synthetic lipopeptide vaccine or primed in vitro with peptide.

Standard synthetic peptide preparations contain numerous peptidic byproducts in small amounts, which may be efficiently recognized by cytotoxic T lymphocytes (CTL). Recognition patterns of such peptide mixtures by CTL may serve as a kind of fingerprint for CTL fine specificity. Three types of H-2Db-restricted CTL were compared in this way. CTL primed in vivo either with A/PR/8/34 influenza virus or with a synthetic lipopeptide vaccine prepared from influenza nucleoprotein (NP) peptide 365-380 showed identical fine specificity. Both recognize virus-infected cells. In contrast, CTL primed in vitro with NP 365-380 had a different fine specificity and they did not recognize virus-infected cells. Most significantly, the two in vivo primed CTL types efficiently recognized the natural viral nonapeptide NP 366-374 presented by virus-infected H-2b cells, whereas the in vitro primed CTL failed to do so.

Efficiency of peptides and lipopeptides for in vivo priming of virus-specific cytotoxic T cells.

Synthetic peptides and a novel type of lipopeptide vaccine, both containing T cell epitopes recognized by Kd-restricted, influenza virus-specific cytotoxic T cells (CTL) were compared in their efficiency to induce virus-specific CTL in vivo. All attempts to induce virus-specific CTL with synthetic peptide (in the absence of adjuvants) failed. However, a latent immunization was observed, resulting in an increased response to the injected peptide seen only after boosting the recipients with immunogenic virus. In contrast, priming with synthetic lipopeptide vaccine (tripalmitoyl-S-glycerylcysteinyl-seryl-serine [P3CSS] coupled to peptide) was successful under most conditions, and matched the priming efficiency seen with infectious virus. The requirements for in vivo priming of virus-specific CTL using lipopeptide suggest that attachment of the lipopeptide to a hydrophobic entity, such as the cell membrane, is responsible for its efficiency.

Identification of naturally processed viral nonapeptides allows their quantification in infected cells and suggests an allele-specific T cell epitope forecast.

Virus-specific cytotoxic T lymphocytes (CTL) recognize virus-derived peptides presented by major histocompatibility complex (MHC) class I molecules on virus-infected cells. Such peptides have been isolated from infected cells and were compared to synthetic peptides. We found previously the Kd- or Db-restricted natural influenza nucleoprotein peptides to coelute on reversed phase high performance liquid chromatography columns with certain peptidic by-products present in synthetic peptide preparations. Here we show by extensive biochemical and immunological comparison that the natural peptides in all respects behave as the surmised synthetic nonapeptides, and thus, must be identical to them. The absolute amounts of these natural peptides contained in infected cells could be determined to be between 220 and 540 copies by comparing with defined amounts of pure synthetic nonapeptides. The comparison of the natural Kd-restricted peptide with published synthetic peptides known to contain other Kd-restricted CTL epitopes suggested a new MHC allele-specific T cell epitope forecast method, based on the defined length of nine amino acid residues and on critical amino acid residues at the second and the last position.

Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells.

Virus-infected cells can be eliminated by cytotoxic T lymphocytes (CTL), which recognize virus-derived peptides bound to major histocompatibility complex (MHC) class I molecules on the cell surface. Until now, this notion has relied on overwhelming but indirect evidence, as the existence of naturally processed viral peptides has not been previously reported. Here we show that such peptides can be extracted from virus-infected cells by acid elution. Both the naturally processed H-2-Db-restricted and H-2-Kd-restricted peptides from influenza nucleoprotein are smaller than the corresponding synthetic peptides, which have first been used to determine the respective CTL epitopes. As with minor histocompatibility antigens, occurrence of viral peptides seems to be heavily dependent on MHC class I molecules, because infected H-2d cells do not contain the H-2-Db-restricted peptide, and infected H-2b cells do not contain the H-2-Kd-restricted peptide. Our data provide direct experimental proof for the above notion on MHC-associated viral peptides on virus-infected cells.

In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine.

Cytotoxic T lymphocytes (CTL) constitute an essential part of the immune response against viral infections. Such CTL recognize peptides derived from viral proteins together with major histocompatibility complex (MHC) class I molecules on the surface of infected cells, and usually require in vivo priming with infectious virus. Here we report that synthetic viral peptides covalently linked to tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS) can efficiently prime influenza-virus-specific CTL in vivo. These lipopeptides are able to induce the same high-affinity CTL as does the infectious virus. Our data are not only relevant to vaccine development, but also have a bearing on basic immune processes leading to the transition of virgin T cells to activated effector cells in vivo, and to antigen presentation by MHC class I molecules.