Optimization of epitope processing enhances immunogenicity of multiepitope DNA vaccines.

Experimental DNA vaccines comprised of multiple minimal cytotoxic T lymphocytes (CTL) epitopes can effectively induce broad CTL responses; however, such constructs frequently exhibit significant variation in epitope immunogenicity. Antigenicity assays utilizing human cells transfected with one such multiepitope construct revealed that the epitopes with poor immunogenicity were inefficiently processed in transfected cells. Compilation of a database of 94 epitope/flanking region combinations, for which immunogenicity was measured experimentally, revealed that the type of residue immediately following the carboxyl-terminus of the epitope exerted a prominent effect on immunogenicity. Experiments utilizing a variety of HBV-specific vaccine constructs demonstrated epitope immunogenicity could be modulated by the insertion of a single amino acid and the effect on immunogenicity could be ascribed to modulation of processing efficiency. These findings demonstrate that multiepitope DNA vaccines can be engineered to enhance CTL immunogenicity by increasing processing efficiency.

Identification and antigenicity of broadly cross-reactive and conserved human immunodeficiency virus type 1-derived helper T-lymphocyte epitopes.

Human immunodeficiency virus (HIV)-specific helper T lymphocytes (HTL) play a key role in the immune control of HIV type 1 (HIV-1) infection, and as such are an important target of potential HIV-1 vaccines. In order to identify HTL epitopes in HIV-1 that might serve as vaccine targets, conserved HIV-1-derived peptides bearing an HLA-DR binding supermotif were tested for binding to a panel of the most representative HLA-DR molecules. Eleven highly cross-reactive binding peptides were identified: three in Gag and eight in Pol. Lymphoproliferative responses to this panel of peptides, as well as to the HIV-1 p24 and p66 proteins, were evaluated with a cohort of 31 HIV-1-infected patients. All 11 peptides were recognized by peripheral blood mononuclear cells from multiple HIV-infected donors. Many of the responsive HIV-infected subjects showed recognition of multiple peptides, indicating that HIV-1-specific T-helper responses may be broadly directed in certain individuals. A strong association existed between recognition of the parental recombinant HIV-1 protein and the corresponding HTL peptides, suggesting that these peptides represent epitopes that are processed and presented during the course of HIV-1 infection. Lastly, responses to the supermotif peptides were mediated by CD4(+) T cells and were restricted by major histocompatibility complex class II molecules. The epitopes described herein are potentially important components of HIV-1 therapeutic and prophylactic vaccines.

Immunization with the HBV core 18-27 epitope elicits CTL responses in humans expressing different HLA-A2 supertype molecules.

The human leukocyte antigen (HLA)-A2 restricted HBV core 18-27 epitope is immunodominant in the context of HLA-A2.1 and subdominant in the context of the other HLA-A2 supertype molecules, as defined by frequency of recognition by memory cytotoxic T lymphocyte (CTL) responses from acute hepatitis B virus (HBV) patients, and on the basis of its binding affinity to purified HLA molecules in vitro. Herein, we show that immunization with a lipopeptide containing HBV core 18-27 epitope induces CTL responses in patients expressing different HLA-A2 supertype molecules, with indistinguishable frequency and magnitude. No difference in responses was noted between patients expressing either one or two different HLA-A2 supertype molecules. Thus, complexes of HBV core 18-27 bound to different HLA-A2 supertype alleles do not appear to act as altered peptide ligands, and do not cross antagonize CTL responses. These results substantiate the immunological relevance of the HLA supertypes concept, and illustrate its potential usefulness for the development of vaccines.

Altered helper T lymphocyte function associated with chronic hepatitis B virus infection and its role in response to therapeutic vaccination in humans.

Theradigm-hepatitis B virus (HBV) is an experimental lipopeptide vaccine designed to stimulate induction of HBV-specific CTL responses in HLA-A2 individuals. Previous studies had demonstrated high immunogenicity in healthy volunteers, but comparatively weak CTL responses in chronically infected HBV patients. Herein, we examined helper T lymphocyte (HTL) responses in chronically infected patients. Despite normal proliferation and IL-2 secretion, IL-12 and IFN-gamma secretion in vitro in response to the vaccine was reduced compared with healthy volunteers. A similar pattern of cytokine secretion was observed following mitogen stimulation, suggesting a general altered balance of Th1/Th2 responses. Further analysis indicated that HTL recall responses to whole tetanus toxoid protein were reduced in chronically infected subjects, and reduced responsiveness correlated with the outcome of Theradigm-HBV immunization. Finally, experiments in HBV transgenic mice indicated that the nonnatural Pan DR HTL epitope, PADRE, is capable of inducing high levels of IFN-gamma secretion and that its inclusion in a lipopeptide incorporating an immunodominant Ld-restricted CTL epitope resulted in breaking tolerance at the CTL level. Overall, our results demonstrate an alteration in the quality of HTL responses induced in chronically infected HBV patients and suggest that use of a potent HTL epitope may be important to overcome CTL tolerance against specific HBV Ags.

The hepatitis B virus-specific CTL responses induced in humans by lipopeptide vaccination are comparable to those elicited by acute viral infection.

We have previously described the development of a lipopeptide-based vaccine, Theradigm-HBV, capable of inducing CTL responses in humans. This vaccine incorporates the HLA-A2.1-restricted CTL epitope hepatitis B core Ag 18-27, linked to the universal helper T lymphocyte (HTL) epitope tetanus toxoid (TT) 830-843. Herein, we report the results of a phase I trial designed to examine the effects of Theradigm-HBV dose and regimen on the magnitude and duration of the memory CTL response. A total of four injections of up to 5 mg/dose were found to be a safe and effective means of generating substantial memory CTL responses. Precursor frequency analysis demonstrated CTL responses of similar magnitude to those previously observed in patients who successfully cleared hepatitis B virus infection and to influenza-specific memory CTL responses induced by natural exposure to influenza virus. Theradigm-HBV induced CTL responses that persisted for more than 9 months after the last injection. HTL responses were associated with significant CTL responses, and sustained HTL activity was necessary for development of persistent memory CTL activity. These results represent the first demonstration of the importance of HTL activity for development of long-lived memory CTL responses in humans. In conclusion, our results show that lipopeptides safely induce specific CTL activity in humans of such magnitude and persistence as to be of potential therapeutic significance.

Polymerase chain reaction cloning of a developmentally regulated member of the sialyltransferase gene family.

We have cloned a new member of the sialyltransferase gene family from newborn rat brain cDNA using the polymerase chain reaction and degenerate primers based on sequences found in a continuous region of homology present in three previously cloned sialyltransferases. The predicted amino acid sequence of the new gene contains 24 of the 31 amino acids that are invariantly conserved in the previously cloned sialyltransferases and encodes a type II transmembrane protein characteristic of glycosyltransferases. In contrast to previously cloned sialyltransferase genes, the expression of the new gene is highly restricted, being abundantly expressed in newborn brain but not in adult brain or any other adult or newborn tissues.

Primary structure of Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase determined by mass spectrometry sequence analysis and molecular cloning. Evidence for a protein motif in the sialyltransferase gene family.

The Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase forms the NeuAc alpha 2,3Gal beta 1,3(4)GlcNAc sequences found in terminal carbohydrate groups of glycoproteins and glycolipids. High energy collision-induced dissociation analysis of tryptic peptides from only 300 pmol of the purified Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase provided 25% of the total amino acid sequence and led to the successful cloning of this enzyme. The peptide sequence information was used to design short degenerate primers for use in the polymerase chain reaction. A long specific cDNA fragment was amplified which was used to isolate a clone from a rat liver cDNA library. The cloned cDNA encodes a 374-amino acid protein containing an amino-terminal signal-anchor sequence characteristic of all cloned glycosyltransferases and produced sialyltransferase activity when transiently expressed in COS-1 cells. When compared with two other cloned sialyltransferases, the primary structure of Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase revealed a homologous region in all three enzymes consisting of a stretch of 55 amino acids located in their catalytic domains. This feature together with lack of homology in the remaining 85% of the sequence of the three sialyltransferases defines a pattern of sequence homology not found in cloned cDNAs of other glycosyltransferase families.

Expression of beta-galactoside alpha 2,6 sialyltransferase blocks synthesis of polysialic acid in Xenopus embryos.

Polysialic acid is a developmentally regulated carbohydrate structure found on neural cell adhesion molecules (NCAM). Expression of beta-galactoside alpha 2,6-sialyltransferase in Xenopus embryos, by injection of mRNA, prevents the polysialylation of NCAM, presumably by introducing a different type of sugar linkage that terminates chain elongation. Abnormalities in neural development result from this treatment, but in general the body plan of the injected embryos is not severely affected. The results provide evidence that the mis-expression of glycosyltransferases can be used to interfere with the normal pattern of glycosylation in whole organisms.

Extended polysialic acid chains (n greater than 55) in glycoproteins from human neuroblastoma cells.

Polysialic acid-containing glycoproteins consisting of extended chains of at least 55 sialyl residues (DP55, where DP represents degree of polymerization) are expressed on human neuroblastoma cells, CHP-134. The strategy used for detecting these unique carbohydrate structures was based on the use of two highly specific prokaryotic-derived enzyme systems and an anti-polysialosyl antibody (H.46). These probes were developed for the detection of polysialic acid on neural cell adhesion molecules (Troy, F. A., Hallenbeck, P. C., McCoy, R. D., and Vimr, E. R. (1987) Methods Enzymol. 138, 169-185). Proof for the presence of long chain multimers of sialic acid was based on two types of experiments which utilized: 1) a glycopeptide fraction of CHP-134 cells, labeled metabolically with D-[3H]GlcN and 2) a membrane fraction from CHP-134 cells which served as an exogenous acceptor of [14C] NeuNAc residues in an Escherichia coli K1 sialyltransferase assay. In vitro, this enzyme CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase catalyzes the transfer of [14C]NeuNAc from CMP-[14C]NeuNAc to exogenous acceptors containing at least 3 sialyl residues. In the first series of experiments, endo-N-acetylneuraminidase (Endo-N), a bacteriophage-derived enzyme specific for hydrolyzing poly-alpha-2,8-sialosyl chains containing a minimum of 5 sialyl residues was used. Limit Endo-N digestion of the 3H-glycopeptides from the [3H] GlcN-labeled cells released short [3H]sialyl oligomers [( 3H]DP1-6) which were degraded to [3H]NeuNAc by exosialidase. Partial Endo-N digestion released a series of [3H]sialyl oligomers extending up to DP55. The longer (DP20-55) and intermediate sized (DP10-20) oligomers were isolated and converted to short oligomers ((3H]DP1-6) by retreating with Endo-N, thus confirming their identity as homo-oligomers of alpha-2,8-linked [3H]NeuNAc residues. In the second series of experiments, a membrane fraction of CHP-134 cells was radiolabeled in vitro with [14C]NeuNAc by E. coli K1 sialyltransferase. The membrane fraction had a major portion of radioactivity that was high Mr and polydisperse (Mr 100,000-250,000) as demonstrated in sodium dodecyl sulfate-polyacrylamide gels. Using Western blotting, pre-existing material of similar size was shown to react with antibody H.46.(ABSTRACT TRUNCATED AT 400 WORDS)