Epitope specificity of murine and human bactericidal antibodies against PorA P1.7,16 induced with experimental meningococcal group B vaccines.

Synthetic peptides derived from the predicted loops 1 and 4 of meningococcal PorA, sero-subtype P1.7,16, were used to study the epitope specificity of murine and human PorA P1.7,16 bactericidal antibodies. The predicted loops 1 and 4 are surface exposed and carry in their apices the sero-subtype epitopes P1.7 (loop 1) or P1.16 (loop 4), respectively. Peptides were synthesized as mono- and multimeric peptides. Murine monoclonal and polyclonal antibodies were induced with meningococcal whole cell preparations. Polyclonal antibodies were evoked in volunteers after one immunization with 50 micrograms or 100 micrograms protein of a hexavalent meningococcal PorA vesicle vaccine. The induction of PorA antibodies was determined in ELISA using purified PorA P1.7,16. The epitope specificity of anti-PorA antibodies for both murine and human antibodies could be demonstrated by direct peptide ELISA using overlapping multimeric peptides almost spanning the entire loops 1 or 4 of the protein. The capacity of peptides to inhibit the bactericidal activity of murine and human antibodies was investigated using meningococcal strain H44/76 (B:15:P1.7,16) as a target strain. Bactericidal activities could be inhibited with both monomeric and multimeric peptides derived from epitopes P1.7 and P1.16.

T-cell responses to outer membrane proteins of Neisseria meningitidis: comparative study of the Opa, Opc, and PorA proteins.

Former studies have shown that the class 5 outer membranes proteins (Opa and Opc proteins) of Neisseria meningitidis are at least as immunogenic as meningococcal porin proteins. High antibody titers to class 5 proteins have been observed in sera obtained during convalescence after meningococcal infection. A strong increase in anti-class 5 antibodies has also been observed in vaccinees who received a meningococcal outer membrane vesicle preparation. The enhanced B-cell response to class 5 proteins may be due to the presence of immunodominant helper T-cell epitopes in these proteins. In order to investigate this hypothesis, we tested purified Opa, Opc, and class 1 proteins for recognition by human T cells. a hierarchy of T-cell immunogenicity was observed among the outer membrane proteins, the Opa protein being more immunogenic than the other proteins. In most cases, the proliferative responses elicited by Opc were higher than the responses observed for the class 1 protein. The epitopes recognized by the immune T cells were identified by using overlapping synthetic peptides spanning the protein sequences of OpaB, Opa5d, and Opc.

Solid-phase synthesis of DNA fragments containing the modified base 7-hydro-8-oxo-2'-deoxyguanosine.

The 5'-(4,4'-dimethoxytrityl) protected 3'-(2-cyanoethoxy)-N,N-diisopropylphosphoramidite of 7-hydro-8-oxo-2'-deoxy-guanosine, the exocyclic amino and lactam functions of which are protected with acetyl and diphenylcarbamoyl groups, respectively, has been prepared from the 8-bromo derivatives of deoxy- and riboguanosine. This synthon, in combination with standard d-nucleoside 3'-(2-cyanoethoxy)-N,N-diisopropylphosphoramidites, was applied successfully to a solid-phase synthesis. Well-defined oligodeoxyribonucleotides containing a 7-hydro-8-oxo-2'-deoxyguanosine residue at predetermined positions were obtained after deprotection with methanolic ammonia and purification by gel filtration.

Human HLA class I- and HLA class II-restricted cloned cytotoxic T lymphocytes identify a cluster of epitopes on the measles virus fusion protein.

The transmembrane fusion (F) glycoprotein of measles virus is an important target antigen of human HLA class I- and class II-restricted cytotoxic T lymphocytes (CTL). Genetically engineered F proteins and nested sets of synthetic peptides spanning the F protein were used to determine sequences of F recognized by a number of F-specific CTL clones. Combined N- and C-terminal deletions of the respective peptides revealed that human HLA class I and HLA class II-restricted CTL efficiently recognize nonapeptides or decapeptides representing epitopes of F. Three distinct sequences recognized by three different HLA class II (DQw1, DR2, and DR4/w53)-restricted CTL clones appear to cluster between amino acids 379 and 466 of F, thus defining an important T-cell epitope area of F. Within this same region, a nonamer peptide of F was found to be recognized by an HLA-B27-restricted CTL clone, as expected on the basis of the structural homology between this peptide and other known HLA-B27 binding peptides.

Solid-phase synthesis and application of double-fluorescent-labeled lipopeptides, containing a CTL-epitope from the measles fusion protein.

The mechanism which enables lipopeptides to induce cytotoxicity is not known. By preparing fluorescent-labeled lipopeptides one might unravel the mechanism of their entry into the cell and their intracellular pathway. A method of preparing double-fluorescent-labeled peptides by solid-phase chemistry is described. As model peptides we have chosen analogs of the sequence RRYPDAVYL, which occurs in the measles fusion protein (F438-446) and is an epitope for cytotoxic T lymphocytes. The peptides Pal-K(TMR)KKKRRYPDAVK(FL)L (7) and Pal-K(FL)KKKRRYPDAVK(TMR)L (8), in which Pal is palmitoyl and K(TMR) and K(FL) are Nepsilon-carboxytetramethylrhodamine- and Nepsilon-carboxyfluorescein-labeled lysyl residues, respectively, were prepared and obtained in approximately 30% yield after purification by high-performance liquid chromatography. The fluorescence of fluorescein and tetramethylrhodamine in lipopeptide Pal-K(TMR)KKKRRYPDAVK(FL)L (7) was quenched to 98-99% due to intramolecular interaction of the labels. On incubation with trypsin (i.e. cleavage at the KKKRR-site) the fluorescence of both labels was restored. The intracellular routing of lipopeptide Pal-K(TMR)KKKRRYPDAVK(FL)L was studied with human melanoma cell line, Mel/J, which was transfected with human leukocyte antigen B*2705. It appeared that the double-fluorescent-labeled lipopeptide was able to induce antigen-specific cytotoxicity. Furthermore, preliminary confocal microscopical studies indicated that this lipopeptide is observed intracellularly.

Simultaneous multiple synthesis and selective conjugation of cyclized peptides derived from a surface loop of a meningococcal class 1 outer membrane protein.

Starting from the alpha-(2,4-dimethoxybenzyl) ester of N-(9-fluorenylmethoxycarbonyl)aspartic acid [Fmoc-Asp-ODmb], side-chain-protected resin-bound Fmoc-peptides containing an N epsilon-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl lysyl [Lys(Dde)] residue were prepared. The C-terminal dimethoxybenzyl esters of aspartic acid were removed with 1% trifluoroacetic acid and 10% anisole in dichloromethane, followed by Fmoc-cleavage in the usual manner. The resin-bound peptides were then cyclized using 1-benzotriazolyloxy-tris-[N-pyrrolidino]phosphonium hexafluorophosphate (PyBOP) in the presence of N-methylmorpholine. The (dimethyldioxocyclohexylidene)ethyl groups of lysine were removed with 1% hydrazine hydrate in N,N-dimethylacetamide, and the liberated side-chain amino functions were modified by reaction with pentafluorophenyl S-acetylmercaptoacetate (SAMA-OPfp). Finally, the peptides were side-chain deprotected, with exception of the Lys(SAMA) residue, and cleaved from the solid support with trifluoroacetic acid/anisole/water, 95/2.5/2.5. Cyclic peptides comprising 7-14 amino acid residues were obtained employing this procedure. As a model conjugation, cyclo[Thr-Asn-Asn-Asn-Leu-Lys(SAMA)-Thr-Lys-Asp] was coupled with bromoacetamide. The same peptide was also coupled with a bromoacetylpeptide to give a well defined peptide/peptide conjugate. All peptides were conjugated to bromoacetylated tetanus toxoid for immunization purposes.

Intraocular T cells of patients with herpes simplex virus (HSV)-induced acute retinal necrosis recognize HSV tegument proteins VP11/12 and VP13/14.

It has previously been shown that T cells specific for the triggering virus infiltrate the eye of patients with herpes simplex virus type 1 (HSV-1)-induced acute retinal necrosis (ARN). The T cells were mainly directed against 0.67-0.73 HSV-1 map region encoded antigens. The fine specificities of genetically different T cell clones (TCC), obtained from affected eyes of 3 patients with HSV-induced ARN and reactive toward this genomic region of HSV-1, were analyzed with recombinant HSV viruses and synthetic peptides. For 1 patient, the HSV-1 UL46 gene encoded tegument protein VP11/12 was identified as the target antigen. Two separate CD4(+) T cell epitopes were defined in VP11/12. TCC from the other 2 patients recognized the HSV-1 UL47 gene encoded tegument protein VP13/14. Two separate CD4(+) VP13/14 T cell epitopes were identified in these patients. Analysis of the data indicates that HSV-1 VP11/12 and VP13/14 are major target antigens for T cells obtained from vitreous fluid samples of the HSV-induced ARN patients studied.

Conjugates of synthetic cyclic peptides elicit bactericidal antibodies against a conformational epitope on a class 1 outer membrane protein of Neisseria meningitidis.

Bactericidal antibodies directed against surface loops of class 1 outer membrane proteins play a crucial role in protection against meningitis and sepsis caused by Neisseria meningitidis. So far, all efforts to obtain protective antibodies against these apparently conformational epitopes by using linear peptide analogs have been in vain. In this study, conjugates of head-to-tail cyclic peptides encompassing the predicted top of a protective surface loop were used for immunization. A series of 18 cyclic peptides with a ring size ranging from 7 to 17 residues, conjugated to tetanus toxoid, was investigated. Antipeptide and anti-whole-cell immunoglobulin G (IgG) titers elicited by the conjugates were determined. Conjugates of three peptides, containing 14, 15, and 17 amino acid residues (peptides 7, 12, and 13, respectively), induced an anti-whole-cell titer when Quillaja saponin A was used as the adjuvant. When alum was used as the adjuvant, the conjugate of peptide 12 did not elicit an anti-whole-cell response. From the Quillaja saponin A group, some of the sera obtained with conjugates of peptides 7 and 12 and all sera obtained with the peptide 13 conjugate were bactericidal in vitro. None of the sera evoked with alum as the adjuvant showed bactericidal activity. Nonbactericidal sera contained IgG1 primarily, whereas bactericidal sera showed significant titers of IgG2a and IgG2b. Class 1 protein-derived synthetic cyclic peptides which are capable of eliciting bactericidal antibodies, such as peptide 13 derived from meningococcal strain H44/76, represent potential candidates for a (semi)synthetic vaccine against meningococcal disease.

Repair and replication of plasmids with site-specific 8-oxodG and 8-AAFdG residues in normal and repair-deficient human cells.

The in vivo mutagenicity of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) and N-(guanin-8-yl)-N-acetyl-2-aminofluorene (8-AAFdG) in human cells was determined by transfecting various cell lines with plasmids that carried a single adduct at a defined site. 8-OxodG is one of the many DNA modifications formed by oxygen radicals, and was found to be highly miscoding during replication with purified DNA polymerases in vitro. Here we show that the frequency of mutations induced by 8-oxodG during replication in vivo is at most only 2% above background. The most predominant mutation found was a single G----T transversion. The frequency of this transversion was found to be 3 to 5-fold increased in excision repair deficient XP-A cells. Interestingly, also the replication of 8-oxodG containing plasmids was significantly impaired (approximately 4-fold) in the XP-A cells, but not in HeLa cells, normal fibroblasts or XP-A revertant cells. When 8-AAFdG containing plasmids were used, the mutation frequencies did not exceed background levels (less than 2%) with any of the cell lines tested. The presence of 8-AAFdG almost completely inhibited plasmid replication (more than 50-fold) in XP-A cells. Apparently, both 8-AAFdG and 8-oxodG are not or poorly repaired in these cells, causing a block of DNA replication. This suggests that both lesions are substrates for excision repair, although to a varying extent.

Role of nucleotide excision repair in processing of O4-alkylthymines in human cells.

O4-Alkylthymines have been implicated as potential carcinogenic DNA lesions. We have studied the effects of O4-methylthymine, O4-ethylthymine, and O4-n-propylthymine in a model system in which a single lesion was located at a defined position on a SV40-based shuttle vector and have found large differences in the effects of these lesions in repair-proficient and nucleotide excision repair-deficient cells. In repair-competent human HeLa cells, normal fibroblasts, and XP-A (2OS) revertant cells, all 3 residues were highly mutagenic; a mutation frequency of approximately 20% was found for both O4-methylthymine and O4-ethylthymine, whereas that of O4-n-propylthymine was approximately 12%. These frequencies were independent of the activity of the O6-alkylguanine DNA alkyltransferase. All three O4-alkylthymines induced T-->C transitions exclusively. In nucleotide excision repair-deficient XP-A cells, however, these lesions were not mutagenic but strongly inhibited plasmid replication (> 90%). These results indicate that O4-alkylthymines are efficiently recognized by the nucleotide excision repair system and cause a complete cessation of plasmid replication if this system is deficient. Nevertheless, proficiency in the nucleotide excision repair pathway correlates with a high frequency of mutation induction by these lesions.

Use of shuttle vectors to study the molecular processing of defined carcinogen-induced DNA damage: mutagenicity of single O4-ethylthymine adducts in HeLa cells.

We developed a simian virus 40 based shuttle vector system to study the molecular consequences of distinct carcinogen-induced DNA lesions in human cells. To establish the mutagenicity of O4-ethylthymine adducts, oligonucleotides carrying a single O4-ethylthymine adduct at a unique position were ligated into the vector molecules. Following replication in HeLa cells on average 23% of the progeny molecules carried a mutation in the region of modification. The vast majority of these mutations represented single T----C transitions at the position of the modified base, most probably as a consequence of mispairing of the O4-ethylthymine residues during replication. To a minor extent the O4-ethylthymine adduct may also induce T----A transversions or double point mutations. The in vivo mutation frequency of the adduct was found to be comparable to that of a C-A mismatch at the same position, but was lower than that expected from in vitro experiments with adducted DNA templates and purified DNA polymerases.